Genetic Mapping with Multiple Levels of Phenotypic Information Reveals Determinants of Lymphocyte Glucocorticoid Sensitivity
2013; Elsevier BV; Volume: 93; Issue: 4 Linguagem: Inglês
10.1016/j.ajhg.2013.08.005
ISSN1537-6605
AutoresJoseph Maranville, Shaneen S. Baxter, David Witonsky, Meredith Chase, Anna Di Rienzo,
Tópico(s)T-cell and B-cell Immunology
ResumoClinical response to glucocorticoids, steroid hormones widely used as pharmaceuticals, varies extensively in that many individuals (∼30%) show a weak response to treatment. Although little is known about the molecular basis of this variation, regulatory polymorphisms are likely to play a key role given that glucocorticoids act largely through activation of a transcription factor, the glucocorticoid receptor. In an effort to characterize the molecular basis of variation in glucocorticoid sensitivity, we measured in vitro lymphocyte glucocorticoid sensitivity and transcriptome-wide response to glucocorticoids in peripheral-blood mononuclear cells from African American healthy donors. We found that variation in lymphocyte glucocorticoid sensitivity was correlated with transcriptional response at 27 genes (false-discovery rate < 0.1). Furthermore, a genome-wide association scan revealed a quantitative trait locus (QTL) for lymphocyte glucocorticoid sensitivity (rs11129354, p = 4 × 10−8); it was also associated with transcriptional response at multiple genes, including many (14/27) where transcriptional response was correlated with lymphocyte glucocorticoid sensitivity. Using allelic-imbalance assays, we show that this QTL is a glucocorticoid-dependent cis-regulatory polymorphism for RBMS3, which encodes an RNA-binding protein known as a tumor suppressor. We found that siRNA-mediated knockdown of RBMS3 expression increased cellular proliferation in PBMCs, consistent with the role of the gene as a negative regulator of proliferation. We propose that differences in lymphocyte glucocorticoid sensitivity reflect variation in transcriptional response, which is influenced by a glucocorticoid-dependent regulatory polymorphism that acts in cis relative to RBMS3 and in trans to affect the transcriptional response of multiple distant genes. Clinical response to glucocorticoids, steroid hormones widely used as pharmaceuticals, varies extensively in that many individuals (∼30%) show a weak response to treatment. Although little is known about the molecular basis of this variation, regulatory polymorphisms are likely to play a key role given that glucocorticoids act largely through activation of a transcription factor, the glucocorticoid receptor. In an effort to characterize the molecular basis of variation in glucocorticoid sensitivity, we measured in vitro lymphocyte glucocorticoid sensitivity and transcriptome-wide response to glucocorticoids in peripheral-blood mononuclear cells from African American healthy donors. We found that variation in lymphocyte glucocorticoid sensitivity was correlated with transcriptional response at 27 genes (false-discovery rate < 0.1). Furthermore, a genome-wide association scan revealed a quantitative trait locus (QTL) for lymphocyte glucocorticoid sensitivity (rs11129354, p = 4 × 10−8); it was also associated with transcriptional response at multiple genes, including many (14/27) where transcriptional response was correlated with lymphocyte glucocorticoid sensitivity. Using allelic-imbalance assays, we show that this QTL is a glucocorticoid-dependent cis-regulatory polymorphism for RBMS3, which encodes an RNA-binding protein known as a tumor suppressor. We found that siRNA-mediated knockdown of RBMS3 expression increased cellular proliferation in PBMCs, consistent with the role of the gene as a negative regulator of proliferation. We propose that differences in lymphocyte glucocorticoid sensitivity reflect variation in transcriptional response, which is influenced by a glucocorticoid-dependent regulatory polymorphism that acts in cis relative to RBMS3 and in trans to affect the transcriptional response of multiple distant genes. Glucocorticoids are endogenous steroid hormones that are also widely used as therapeutic agents to treat a variety of diseases, such as asthma,1Verini M. Rossi N. Dalfino T. Verrotti A. Di Gioacchino M. Chiarelli F. Lack of correlation between clinical patterns of asthma and airway obstruction.Allergy Asthma Proc. 2001; 22: 297-302PubMed Google Scholar, 2NHLBIHighlights of the Expert Panel Report 2: Guidelines for Diagnosis and Management of Asthma. NIH Publications, Bethesda1997: 50Google Scholar, 3Chung K.F. O'Byrne P. Pharmacological agents used to treat asthma.in: Fabbri L.M. Asthma. 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Bull. 2000; 56: 1054-1070Crossref PubMed Scopus (283) Google Scholar Candidate-gene studies have also identified associations with glucocorticoid response in individuals with asthma, but these polymorphisms explain little of the common variation in glucocorticoid response and/or are unreliably associated with response (reviewed in Tantisira et al.22). In an effort to move beyond candidate genes, several recent studies have performed genome-wide association mapping of glucocorticoid response in individuals with asthma, revealing associations at a cis-regulatory polymorphism for GLCCI122Tantisira K.G. Lasky-Su J. Harada M. Murphy A. Litonjua A.A. Himes B.E. Lange C. Lazarus R. Sylvia J. Klanderman B. et al.Genomewide association between GLCCI1 and response to glucocorticoid therapy in asthma.N. Engl. J. Med. 2011; 365: 1173-1183Crossref PubMed Scopus (285) Google Scholar (MIM 614283) and a polymorphism in T23Tantisira K.G. Damask A. Szefler S.J. Schuemann B. Markezich A. Su J. 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Research Blood Components requires that all donors sign an institutional-review-board-approved consent form giving permission for the collection, use, and/or sale of blood for research purposes. Blood samples were not shipped to the University of Chicago with individually identifiable information. PBMCs were cultured at 2 × 105 cells per well in 10% charcoal-stripped media in 96-well plates and treated in triplicate (the median value was used) with phytohemagglutinin (PHA) (2.5 μg/ml) and either a vehicle (EtOH) or dexamethasone (dex) (1 μM, 0.1 μM, 10 nM, or 1 nM). H3-thymidine incorporation was used for measuring cell proliferation after 48 hr of treatment. Lymphocyte glucocorticoid sensitivity has been shown to be correlated with clinical response to glucocorticoid therapy in individuals with a wide range of diseases, including asthma,24Corrigan C.J. Bungre J.K. Assoufi B. Cooper A.E. Seddon H. Kay A.B. 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Apoptosis of lymphocytes induced by glucocorticoids and relationship to therapeutic efficacy in patients with systemic lupus erythematosus.Arthritis Rheum. 1998; 41: 823-830Crossref PubMed Scopus (74) Google Scholar ulcerative colitis,31Hearing S.D. Norman M. Smyth C. Foy C. Dayan C.M. Wide variation in lymphocyte steroid sensitivity among healthy human volunteers.J. Clin. Endocrinol. Metab. 1999; 84: 4149-4154Crossref PubMed Scopus (88) Google Scholar and renal transplant rejection,32Langhoff E. Ladefoged J. Jakobsen B.K. Platz P. Ryder L.P. Svejgaard A. Thaysen J.H. Recipient lymphocyte sensitivity to methylprednisolone affects cadaver kidney graft survival.Lancet. 1986; 1: 1296-1297Abstract PubMed Scopus (84) Google Scholar as well as with risk of developing posttraumatic stress disorder.33International HapMap ConsortiumThe International HapMap Project.Nature. 2003; 426: 789-796Crossref PubMed Scopus (4969) Google Scholar The reported correlations between in vitro lymphocyte glucocorticoid sensitivity and clinical response in diseases affecting different tissues suggest that glucocorticoid nonresponsiveness is mediated by lymphocytes or that the in vitro assay captures shared mechanisms of glucocorticoid action across cell types, making this an appropriate experimental system for investigating the molecular basis of glucocorticoid resistance. Interestingly, patterns of variation in in vitro lymphocyte glucocorticoid sensitivity among individuals with diseases that are treated with glucocorticoids are similar to those observed in healthy donors: they have similar proportions of nonresponsive individuals and similar interethnic differences in response.13Maranville J.C. Baxter S.S. Torres J.M. Di Rienzo A. Inter-ethnic differences in lymphocyte sensitivity to glucocorticoids reflect variation in transcriptional response.Pharmacogenomics J. 2013; 13: 121-129Crossref PubMed Scopus (22) Google Scholar, 31Hearing S.D. Norman M. Smyth C. Foy C. Dayan C.M. Wide variation in lymphocyte steroid sensitivity among healthy human volunteers.J. Clin. Endocrinol. Metab. 1999; 84: 4149-4154Crossref PubMed Scopus (88) Google Scholar, 34Federico M.J. Covar R.A. Brown E.E. Leung D.Y. Spahn J.D. Racial differences in T-lymphocyte response to glucocorticoids.Chest. 2005; 127: 571-578Crossref PubMed Scopus (61) Google Scholar This suggests that glucocorticoid sensitivity is a property of the general population and manifests itself as clinical resistance when individuals develop a disease for which glucocorticoids are administered. Here, we investigate the molecular basis of variation in in vitro lymphocyte glucocorticoid sensitivity, allowing us to explore general mechanisms of glucocorticoid resistance without limiting ourselves to a particular disease or tissue. We note, however, that this approach cannot identify glucocorticoid-resistance mechanisms that are specific to other cell types or other experimental conditions. Although a variety of metrics (e.g., IC50, a measure of potency) have been correlated with clinical response,24Corrigan C.J. Bungre J.K. Assoufi B. Cooper A.E. Seddon H. Kay A.B. 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Glucocorticoid pharmacokinetics, glucocorticoid receptor characteristics, and inhibition of peripheral blood T cell proliferation by glucocorticoids in vitro.Am. Rev. Respir. Dis. 1991; 144: 1016-1025Crossref PubMed Scopus (231) Google Scholar, 28Poznansky M.C. Gordon A.C. Douglas J.G. Krajewski A.S. Wyllie A.H. Grant I.W. Resistance to methylprednisolone in cultures of blood mononuclear cells from glucocorticoid-resistant asthmatic patients.Clin. Sci. 1984; 67: 639-645PubMed Google Scholar, 29Kirkham B.W. Corkill M.M. Davison S.C. Panayi G.S. Response to glucocorticoid treatment in rheumatoid arthritis: in vitro cell mediated immune assay predicts in vivo responses.J. Rheumatol. 1991; 18: 821-825PubMed Google Scholar, 30Seki M. Ushiyama C. Seta N. Abe K. Fukazawa T. Asakawa J. Takasaki Y. Hashimoto H. Apoptosis of lymphocytes induced by glucocorticoids and relationship to therapeutic efficacy in patients with systemic lupus erythematosus.Arthritis Rheum. 1998; 41: 823-830Crossref PubMed Scopus (74) Google Scholar, 31Hearing S.D. Norman M. Smyth C. Foy C. Dayan C.M. Wide variation in lymphocyte steroid sensitivity among healthy human volunteers.J. Clin. Endocrinol. Metab. 1999; 84: 4149-4154Crossref PubMed Scopus (88) Google Scholar, 32Langhoff E. Ladefoged J. Jakobsen B.K. Platz P. Ryder L.P. Svejgaard A. Thaysen J.H. Recipient lymphocyte sensitivity to methylprednisolone affects cadaver kidney graft survival.Lancet. 1986; 1: 1296-1297Abstract PubMed Scopus (84) Google Scholar, 33International HapMap ConsortiumThe International HapMap Project.Nature. 2003; 426: 789-796Crossref PubMed Scopus (4969) Google Scholar percent inhibition at a high dose (e.g., Imax, a measure of efficacy) was previously shown to be particularly strongly correlated.31Hearing S.D. Norman M. Smyth C. Foy C. Dayan C.M. Wide variation in lymphocyte steroid sensitivity among healthy human volunteers.J. Clin. Endocrinol. Metab. 1999; 84: 4149-4154Crossref PubMed Scopus (88) Google Scholar We measured both Imax and IC50 in each donor. We found that age, gender, circulating cortisol levels (catalog no. 11875116160, Roche Diagnostics), baseline GR transcript levels, and basal PBMC proliferation levels were not significantly correlated with Imax or IC50, consistent with previous results.13Maranville J.C. Baxter S.S. Torres J.M. Di Rienzo A. Inter-ethnic differences in lymphocyte sensitivity to glucocorticoids reflect variation in transcriptional response.Pharmacogenomics J. 2013; 13: 121-129Crossref PubMed Scopus (22) Google Scholar, 31Hearing S.D. Norman M. Smyth C. Foy C. Dayan C.M. Wide variation in lymphocyte steroid sensitivity among healthy human volunteers.J. Clin. Endocrinol. Metab. 1999; 84: 4149-4154Crossref PubMed Scopus (88) Google Scholar We also measured PBMC composition in each donor by using flow cytometry with the following antibodies: anti-CD3-PE-Cy7 to mark T cells (BDB560910), anti-CD14-FITC to mark monocytes (NC0088365), anti-CD20-PE to mark B cells (BDB555623), anti-CD4-PerCP-Cy5.5 to mark T helper cells (BDB560650), and anti-CD8-APC to mark cytotoxic T cells (COIM2469). All antibodies were obtained from Fisher Scientific. We found a significant and negative correlation between Imax and the proportion of CD8+ T cells (p = 0.024). This is consistent with clinical studies that show that CD8+ T cell proportions are correlated with insensitivity to glucocorticoid treatment in individuals with multiple myeloma35Koike M. Sekigawa I. Okada M. Matsumoto M. Iida N. Hashimoto H. Oshimi K. Relationship between CD4(+)/CD8(+) T cell ratio and T cell activation in multiple myeloma: reference to IL-16.Leuk. Res. 2002; 26: 705-711Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar and systematic lupus erythematosus.36Matsushita M. Hayashi T. Ando S. Sekigawa I. Iida N. Hashimoto H. Hirose S. Changes of CD4/CD8 ratio and interleukin-16 in systemic lupus erythematosus.Clin. Rheumatol. 2000; 19: 270-274Crossref PubMed Scopus (17) Google Scholar Both Imax and IC50 were corrected for all covariates above in all subsequent analyses. To interrogate the role of transcriptional response in variable glucocorticoid sensitivity, we profiled gene expression in the presence of either dex or vehicle after 6 hr of treatment in PHA-treated PBMCs from 85 of the same 88 healthy donors assayed, in parallel, for lymphocyte glucocorticoid sensitivity. PBMCs from each donor were grown in 24-well plates (106 cells per well) and treated with PHA (2.5 μg/ml) and either vehicle (EtOH) or dex (1 μM) in three replicates that were pooled before RNA extraction. Total RNA was extracted from each pool with the RNeasy Plus Mini Kit (QIAGEN 74134). Expression was profiled with Illumina HumanHT-12 v4 Expression BeadChips. Low-level microarray analysis was performed with the Bioconductor software package LUMI37Du P. Kibbe W.A. Lin S.M. lumi: a pipeline for processing Illumina microarray.Bioinformatics. 2008; 24: 1547-1548Crossref PubMed Scopus (1621) Google Scholar in R, including variance-stabilizing transformation,38Lin S.M. Du P. Huber W. Kibbe W.A. Model-based variance-stabilizing transformation for Illumina microarray data.Nucleic Acids Res. 2008; 36: e11Crossref PubMed Scopus (402) Google Scholar filtering based on expression levels, and quantile normalization across all arrays. Probes were annotated by mapping to the mRNA sequences from RefSeq with the use of BLAT, and probes that mapped to multiple genes or that contained one or more HapMap SNPs were excluded from further analyses. The microarray data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE48801. We used linear regression to identify genes that were differentially expressed between dex- and vehicle-treated samples and controlled for potential covariates (i.e., batch, age, gender, cell proportions, time of blood draw, and cortisol levels). Similarly to previous studies of glucocorticoid response in PBMCs13Maranville J.C. Baxter S.S. Torres J.M. Di Rienzo A. Inter-ethnic differences in lymphocyte sensitivity to glucocorticoids reflect variation in transcriptional response.Pharmacogenomics J. 2013; 13: 121-129Crossref PubMed Scopus (22) Google Scholar, 14Hakonarson H. Bjornsdottir U.S. Halapi E. Bradfield J. Zink F. Mouy M. Helgadottir H. Gudmundsdottir A.S. Andrason H. Adalsteinsdottir A.E. et al.Profiling of genes expressed in peripheral blood mononuclear cells predicts glucocorticoid sensitivity in asthma patients.Proc. Natl. Acad. Sci. 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USA. 2003; 100: 9440-9445Crossref PubMed Scopus (7083) Google Scholar in R.) To assess the effect of PHA stimulation on the transcriptome, we also compared gene expression between PHA-treated and PHA-untreated PBMCs from 9 of the 85 donors. Because we measured expression in the absence of PHA in a much smaller set of samples, we used a hierarchical model to stabilize estimates of the variance (LIMMA42Smyth G.K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments.Stat. Appl. Genet. Mol. Biol. 2004; 3: e3Google Scholar in R) to identify genes differentially expressed between PHA- and blank-treated samples. We found that 3,640 genes were differentially expressed (FDR < 0.01, Figure S1) after PHA stimulation. We used the Database for Annotation, Visualization, and Integrated Discovery (DAVID)43Huang W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4: 44-57Crossref PubMed Scopus (25346) Google Scholar, 44Dennis Jr., G. Sherman B.T. Hosack D.A. Yang J. Gao W. Lane H.C. Lempicki R.A. DAVID: Database for Annotation, Visualization, and Integrated Discovery.Genome Biol. 2003; 4: 3Crossref PubMed Google Scholar to identify gene sets enriched among differentially expressed genes (by using all genes expressed in PBMCs as a background), which revealed that genes upregulated in PHA-stimulated PBMCs were enriched with gene sets consistent with the proinflammatory properties of PHA (Table S1); these gene sets included pathways related to both adaptive (e.g., "regulation of T cell activation," p = 5.4 × 10−9, FDR = 1.7 × 10−6) and innate (e.g., "innate immune response," p = 1.9 × 10−4, FDR = 8.8 × 10−3) immune response. Downregulated genes were enriched with biological processes related to cell death (e.g., "lysosome," p = 3.3 × 10−6, FDR = 5.9 × 10−4, Table S2). Consistent with dex inhibition of PHA-mediated cell proliferation, 3,000 of the 3,640 genes differentially expressed in the presence of PHA (FDR < 0.01) were also differentially expressed after dex treatment (FDR < 0.01). Consistent with the inhibitory effect of glucocorticoids on PHA-mediated cell proliferation, dex treatment largely reversed the transcriptional effects of PHA: considering only the genes differentially expressed in both treatments (FDR < 0.01), 91.7% of genes upregulated by PHA were downregulated by glucocorticoids and 88.1% of genes downregulated by PHA were upregulated after glucocorticoid treatment (Figure S1). We found that genes that were differentially expressed by PHA treatment (FDR < 0.01) and not differentially expressed by glucocorticoid treatment (p > 0.05) were significantly enriched with the Panther glycolysis pathway (p = 4.1 × 10−4, FDR = 0.037). PHA is known to increase glycolysis in lymphocytes,45Cordiali-Fei P. Floridi A. Apollonj Ghetti M.C. Natali P.G. Estimation of PHA induced transformation in peripheral blood lymphocytes through measurement of their increased glycolysis. An enzymatic micromethod.Immunol. Commun. 1980; 9: 389-401PubMed Google Scholar a
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