Peripheral blood gene expression patterns discriminate among chronic inflammatory diseases and healthy controls and identify novel targets

Bertalan Mesko, Szilard Poliska, Andrea Szegedi, Zoltan Szekanecz, Karoly Palatka, Maria Papp, Laszlo Nagy, Bertalan Mesko, Szilard Poliska, Andrea Szegedi, Zoltan Szekanecz, Karoly Palatka, Maria Papp, Laszlo Nagy

Abstract

Background: Chronic inflammatory diseases including inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis), psoriasis and rheumatoid arthritis (RA) afflict millions of people worldwide, but their pathogenesis is still not well understood. It is also not well known if distinct changes in gene expression characterize these diseases and if these patterns can discriminate between diseased and control patients and/or stratify the disease. The main focus of our work was the identification of novel markers that overlap among the 3 diseases or discriminate them from each other.

Methods: Diseased (n = 13, n = 15 and n = 12 in IBD, psoriasis and RA respectively) and healthy patients (n = 18) were recruited based on strict inclusion and exclusion criteria; peripheral blood samples were collected by clinicians (30 ml) in Venous Blood Vacuum Collection Tubes containing EDTA and peripheral blood mononuclear cells were separated by Ficoll gradient centrifugation. RNA was extracted using Trizol reagent. Gene expression data was obtained using TaqMan Low Density Array (TLDA) containing 96 genes that were selected by an algorithm and the statistical analyses were performed in Prism by using non-parametric Mann-Whitney U test (P-values < 0.05).

Results: Here we show that using a panel of 96 disease associated genes and measuring mRNA expression levels in peripheral blood derived mononuclear cells; we could identify disease-specific gene panels that separate each disease from healthy controls. In addition, a panel of five genes such as ADM, AQP9, CXCL2, IL10 and NAMPT discriminates between all samples from patients with chronic inflammation and healthy controls. We also found genes that stratify the diseases and separate different subtypes or different states of prognosis in each condition.

Conclusions: These findings and the identification of five universal markers of chronic inflammation suggest that these diseases have a common background in pathomechanism, but still can be separated by peripheral blood gene expression. Importantly, the identified genes can be associated with overlapping biological processes including changed inflammatory response. Gene panels based on such markers can play a major role in the development of personalized medicine, in monitoring disease progression and can lead to the identification of new potential drug targets in chronic inflammation.

Figures

Figure 1
Figure 1
Flowchart of gene selection process. A flow chart of the gene selection process shows the steps through which we chose the final 96 genes out of the database containing 400 genes.
Figure 2
Figure 2
Fold changes of genes differentiating between diseased and control samples. Fold change values of genes, showing statistically significant (Mann-Whitney U test) differential expression between diseased and control patients, were generated from RT-QPCR measurements and represent the difference of the means of the diseased and control groups. Principal component analysis was performed and separates the two groups of samples. 2a represents the IBD, 2b the psoriasis and 2c the RA gene panel.
Figure 3
Figure 3
Genes separating chronic inflammatory diseases from controls. (a) Venn diagram shows all of those genes that show significant differences between each disease group and control samples. Each set contains genes that separate control samples only from the particular disease or diseases. Underlined genes were down-regulated compared to healthy controls. (b) Gene interaction analysis in GeneSpring GX in Direct Interactions mode highlights 28 genes that have direct interactions with each other while 25 genes have no direct interactions. Genes with the highest number of interactions are shown in extended size. Cyclooxygenase 2 enzyme is located in the middle of this pathway network. The genes that showed significant differences between one of the diseased groups and healthy controls have color codes in which blue represents RA, green represents IBD and red codes for psoriasis-related genes.
Figure 4
Figure 4
Functional categorization of significantly changing genes. We found 53 genes that show significantly differences between chronic inflammatory diseases and control samples. These genes were grouped into functional categories made by EASE. Genes were grouped in the category that had the highest relevance according to the EASE software. Colors represent disease groups. The size of the diagrams correlates with the number of genes in each set. Overlapping sets of genes are shown as overlapping diagrams.
Figure 5
Figure 5
Correlation between gene expression levels and clinical parameters. Normalized mRNA levels of genes showing significant differences between patients with (a) Crohn's disease and Ulcerative colitis; (b) psoriasis patients with and without arthritis; (c) RA patients with and without MR-confirmed bone erosion were generated from RT-QPCR measurements and represent the means of the expression levels of the diseased and control groups.
Figure 6
Figure 6
Peripheral blood derived universal markers of chronic inflammation. Normalized mRNA levels of genes (with SD) showing significant differences between all the samples of chronic inflammatory diseases and healthy controls were generated from RT-QPCR measurements.
Figure 7
Figure 7
Pathway analysis of universal markers of chronic inflammation. Pathway analysis was carried out with GeneSpring GX Biological Processes mode that revealed all the biological processes the genes are related to. Circles represent the 5 genes; deltoids represent biological processes.

References

    1. Silman AJ, Pearson JE. Epidemiology and genetics of rheumatoid arthritis. Arthritis Res. 2002;4(Suppl 3):S265–272. doi: 10.1186/ar578.
    1. Langley RG, Krueger GG, Griffiths CE. Psoriasis: epidemiology, clinical features, and quality of life. Ann Rheum Dis. 2005;64(Suppl 2):ii18–23. doi: 10.1136/ard.2004.033217. discussion ii24-15.
    1. Loftus EV Jr. Clinical epidemiology of inflammatory bowel disease: Incidence, prevalence, and environmental influences. Gastroenterology. 2004;126(6):1504–1517. doi: 10.1053/j.gastro.2004.01.063.
    1. Wu F, Dassopoulos T, Cope L, Maitra A, Brant SR, Harris ML, Bayless TM, Parmigiani G, Chakravarti S. Genome-wide gene expression differences in Crohn's disease and ulcerative colitis from endoscopic pinch biopsies: insights into distinctive pathogenesis. Inflamm Bowel Dis. 2007;13(7):807–821. doi: 10.1002/ibd.20110.
    1. Nomura I, Gao B, Boguniewicz M, Darst MA, Travers JB, Leung DY. Distinct patterns of gene expression in the skin lesions of atopic dermatitis and psoriasis: a gene microarray analysis. J Allergy Clin Immunol. 2003;112(6):1195–1202. doi: 10.1016/j.jaci.2003.08.049.
    1. van Baarsen LG, Wijbrandts CA, Timmer TC, Pouw Kraan TC van der, Tak PP, Verweij CL. Synovial tissue heterogeneity in rheumatoid arthritis in relationship to disease activity and biomarkers in peripheral blood. Arthritis Rheum. 2010. in press .
    1. Aziz H, Zaas A, Ginsburg GS. Peripheral blood gene expression profiling for cardiovascular disease assessment. Genomic Med. 2007;1(3-4):105–112. doi: 10.1007/s11568-008-9017-x.
    1. Ma XJ, Salunga R, Tuggle JT, Gaudet J, Enright E, McQuary P, Payette T, Pistone M, Stecker K, Zhang BM. Gene expression profiles of human breast cancer progression. Proc Natl Acad Sci USA. 2003;100(10):5974–5979. doi: 10.1073/pnas.0931261100.
    1. Maas K, Chan S, Parker J, Slater A, Moore J, Olsen N, Aune TM. Cutting edge: molecular portrait of human autoimmune disease. J Immunol. 2002;169(1):5–9.
    1. Koczan D, Guthke R, Thiesen HJ, Ibrahim SM, Kundt G, Krentz H, Gross G, Kunz M. Gene expression profiling of peripheral blood mononuclear leukocytes from psoriasis patients identifies new immune regulatory molecules. Eur J Dermatol. 2005;15(4):251–257.
    1. Junta CM, Sandrin-Garcia P, Fachin-Saltoratto AL, Mello SS, Oliveira RD, Rassi DM, Giuliatti S, Sakamoto-Hojo ET, Louzada-Junior P, Donadi EA. Differential gene expression of peripheral blood mononuclear cells from rheumatoid arthritis patients may discriminate immunogenetic, pathogenic and treatment features. Immunology. 2009;127(3):365–372. doi: 10.1111/j.1365-2567.2008.03005.x.
    1. Burczynski ME, Peterson RL, Twine NC, Zuberek KA, Brodeur BJ, Casciotti L, Maganti V, Reddy PS, Strahs A, Immermann F. Molecular classification of Crohn's disease and ulcerative colitis patients using transcriptional profiles in peripheral blood mononuclear cells. J Mol Diagn. 2006;8(1):51–61. doi: 10.2353/jmoldx.2006.050079.
    1. Steinbach D, Schramm A, Eggert A, Onda M, Dawczynski K, Rump A, Pastan I, Wittig S, Pfaffendorf N, Voigt A. Identification of a set of seven genes for the monitoring of minimal residual disease in pediatric acute myeloid leukemia. Clin Cancer Res. 2006;12(8):2434–2441. doi: 10.1158/1078-0432.CCR-05-2552.
    1. Schobesberger M, Baltzer A, Oberli A, Kappeler A, Gugger M, Burger H, Jaggi R. Gene expression variation between distinct areas of breast cancer measured from paraffin-embedded tissue cores. BMC Cancer. 2008;8:343. doi: 10.1186/1471-2407-8-343.
    1. Swingler TE, Waters JG, Davidson RK, Pennington CJ, Puente XS, Darrah C, Cooper A, Donell ST, Guile GR, Wang W. Degradome expression profiling in human articular cartilage. Arthritis Res Ther. 2009;11(3):R96. doi: 10.1186/ar2741.
    1. Kosa JP, Balla B, Kiss J, Podani J, Takacs I, Lazary A, Nagy Z, Bacsi K, Karsai A, Speer G. Postmenopausal expression changes of immune system-related genes in human bone tissue. J Clin Immunol. 2009;29(6):761–768. doi: 10.1007/s10875-009-9321-9.
    1. Hosack DA, Dennis G Jr, Sherman BT, Lane HC, Lempicki RA. Identifying biological themes within lists of genes with EASE. Genome Biol. 2003;4(10):R70. doi: 10.1186/gb-2003-4-10-r70.
    1. Dudley JT, Tibshirani R, Deshpande T, Butte AJ. Disease signatures are robust across tissues and experiments. Mol Syst Biol. 2009;5:307. doi: 10.1038/msb.2009.66.
    1. Batliwalla FM, Baechler EC, Xiao X, Li W, Balasubramanian S, Khalili H, Damle A, Ortmann WA, Perrone A, Kantor AB. Peripheral blood gene expression profiling in rheumatoid arthritis. Genes Immun. 2005;6(5):388–397. doi: 10.1038/sj.gene.6364209.
    1. Barnes MG, Aronow BJ, Luyrink LK, Moroldo MB, Pavlidis P, Passo MH, Grom AA, Hirsch R, Giannini EH, Colbert RA. Gene expression in juvenile arthritis and spondyloarthropathy: pro-angiogenic ELR+ chemokine genes relate to course of arthritis. Rheumatology (Oxford) 2004;43(8):973–979. doi: 10.1093/rheumatology/keh224.
    1. Gu J, Marker-Hermann E, Baeten D, Tsai WC, Gladman D, Xiong M, Deister H, Kuipers JG, Huang F, Song YW. A 588-gene microarray analysis of the peripheral blood mononuclear cells of spondyloarthropathy patients. Rheumatology (Oxford) 2002;41(7):759–766. doi: 10.1093/rheumatology/41.7.759.
    1. Bruneau N, Richard S, Silvy F, Verine A, Lombardo D. Lectin-like Ox-LDL receptor is expressed in human INT-407 intestinal cells: involvement in the transcytosis of pancreatic bile salt-dependent lipase. Mol Biol Cell. 2003;14(7):2861–2875. doi: 10.1091/mbc.E02-08-0544.
    1. Dwinell MB, Eckmann L, Leopard JD, Varki NM, Kagnoff MF. Chemokine receptor expression by human intestinal epithelial cells. Gastroenterology. 1999;117(2):359–367. doi: 10.1053/gast.1999.0029900359.
    1. Nagaishi T, Chen Z, Chen L, Iijima H, Nakajima A, Blumberg RS. CEACAM1 and the regulation of mucosal inflammation. Mucosal Immunol. 2008;1(Suppl 1):S39–42. doi: 10.1038/mi.2008.50.
    1. Fireman E, Kraiem Z, Sade O, Greif J, Fireman Z. Induced sputum-retrieved matrix metalloproteinase 9 and tissue metalloproteinase inhibitor 1 in granulomatous diseases. Clin Exp Immunol. 2002;130(2):331–337. doi: 10.1046/j.1365-2249.2002.t01-1-02001.x.
    1. Garg P, Vijay-Kumar M, Wang L, Gewirtz AT, Merlin D, Sitaraman SV. Matrix metalloproteinase-9-mediated tissue injury overrides the protective effect of matrix metalloproteinase-2 during colitis. Am J Physiol Gastrointest Liver Physiol. 2009;296(2):G175–184. doi: 10.1152/ajpgi.90454.2008.
    1. Gonsky R, Deem RL, Targan SR. Distinct Methylation of IFNG in the Gut. J Interferon Cytokine Res. 2009;29(7):407–414. doi: 10.1089/jir.2008.0109.
    1. Hendel J, Nielsen OH. Expression of cyclooxygenase-2 mRNA in active inflammatory bowel disease. Am J Gastroenterol. 1997;92(7):1170–1173.
    1. Rottman JB, Smith TL, Ganley KG, Kikuchi T, Krueger JG. Potential role of the chemokine receptors CXCR3, CCR4, and the integrin alphaEbeta7 in the pathogenesis of psoriasis vulgaris. Lab Invest. 2001;81(3):335–347.
    1. Koczan D, Drynda S, Hecker M, Drynda A, Guthke R, Kekow J, Thiesen HJ. Molecular discrimination of responders and nonresponders to anti-TNF alpha therapy in rheumatoid arthritis by etanercept. Arthritis Res Ther. 2008;10(3):R50.
    1. Voort R van der, van Lieshout AW, Toonen LW, Sloetjes AW, Berg WB van den, Figdor CG, Radstake TR, Adema GJ. Elevated CXCL16 expression by synovial macrophages recruits memory T cells into rheumatoid joints. Arthritis Rheum. 2005;52(5):1381–1391. doi: 10.1002/art.21004.
    1. Yang MH, Wu FX, Xie CM, Qing YF, Wang GR, Guo XL, Tang Z, Zhou JG, Yuan GH. Expression of CC chemokine ligand 5 in patients with rheumatoid arthritis and its correlation with disease activity and medication. Chin Med Sci J. 2009;24(1):50–54. doi: 10.1016/S1001-9294(09)60059-6.
    1. Kehlen A, Pachnio A, Thiele K, Langner J. Gene expression induced by interleukin-17 in fibroblast-like synoviocytes of patients with rheumatoid arthritis: upregulation of hyaluronan-binding protein TSG-6. Arthritis Res Ther. 2003;5(4):R186–192. doi: 10.1186/ar762.
    1. Zuo XX, Zhou YO, Gong YH, Wang YP, Tang DL, Xiao XZ. [Expression of high mobility group box chromosomal protein 1 in peripheral blood of patients with rheumatoid arthritis] Zhonghua Nei Ke Za Zhi. 2007;46(7):547–550.
    1. Zini N, Lisignoli G, Solimando L, Bavelloni A, Grassi F, Guidotti L, Trimarchi C, Facchini A, Maraldi NM. IL1-beta and TNF-alpha induce changes in the nuclear polyphosphoinositide signalling system in osteoblasts similar to that occurring in patients with rheumatoid arthritis: an immunochemical and immunocytochemical study. Histochem Cell Biol. 2003;120(3):243–250. doi: 10.1007/s00418-003-0563-y.
    1. Schulze-Koops H, Davis LS, Kavanaugh AF, Lipsky PE. Elevated cytokine messenger RNA levels in the peripheral blood of patients with rheumatoid arthritis suggest different degrees of myeloid cell activation. Arthritis Rheum. 1997;40(4):639–647. doi: 10.1002/art.1780400408.
    1. Alexander RJ, Panja A, Kaplan-Liss E, Mayer L, Raicht RF. Expression of protooncogene-encoded mRNA by colonic epithelial cells in inflammatory bowel disease. Dig Dis Sci. 1996;41(4):660–669. doi: 10.1007/BF02213120.
    1. Pouw Kraan TC van der, Zwiers A, Mulder CJ, Kraal G, Bouma G. Acute experimental colitis and human chronic inflammatory diseases share expression of inflammation-related genes with conserved Ets2 binding sites. Inflamm Bowel Dis. 2009;15(2):224–235. doi: 10.1002/ibd.20747.
    1. Haas SL, Abbatista M, Brade J, Singer MV, Bocker U. Interleukin-18 serum levels in inflammatory bowel diseases: correlation with disease activity and inflammatory markers. Swiss Med Wkly. 2009;139(9-10):140–145.
    1. Mitsuyama K, Sata M, Rose-John S. Interleukin-6 trans-signaling in inflammatory bowel disease. Cytokine Growth Factor Rev. 2006;17(6):451–461. doi: 10.1016/j.cytogfr.2006.09.003.
    1. Hitchon CA, Danning CL, Illei GG, El-Gabalawy HS, Boumpas DT. Gelatinase expression and activity in the synovium and skin of patients with erosive psoriatic arthritis. J Rheumatol. 2002;29(1):107–117.
    1. Partsch G, Wagner E, Leeb BF, Broll H, Dunky A, Smolen JS. T cell derived cytokines in psoriatic arthritis synovial fluids. Ann Rheum Dis. 1998;57(11):691–693. doi: 10.1136/ard.57.11.691.
    1. Chang X, Zhao Y, Sun S, Zhang Y, Zhu Y. The expression of PADI4 in synovium of rheumatoid arthritis. Rheumatol Int. 2009;29(12):1411–6. doi: 10.1007/s00296-009-0870-2.
    1. Li Y, Begovich AB. Unraveling the genetics of complex diseases: Susceptibility genes for rheumatoid arthritis and psoriasis. Semin Immunol. 2009;21(6):318–27. doi: 10.1016/j.smim.2009.04.002.
    1. Marutsuka K, Nawa Y, Asada Y, Hara S, Kitamura K, Eto T, Sumiyoshi A. Adrenomedullin and proadrenomudullin N-terminal 20 peptide (PAMP) are present in human colonic epithelia and exert an antimicrobial effect. Exp Physiol. 2001;86(5):543–545. doi: 10.1113/eph8602250.
    1. Chosa E, Hamada H, Kitamura K, Eto T, Tajima N. Increased plasma and joint tissue adrenomedullin concentrations in patients with rheumatoid arthritis compared to those with osteoarthritis. J Rheumatol. 2003;30(12):2553–2556.
    1. Scaife S, Brown R, Kellie S, Filer A, Martin S, Thomas AM, Bradfield PF, Amft N, Salmon M, Buckley CD. Detection of differentially expressed genes in synovial fibroblasts by restriction fragment differential display. Rheumatology(Oxford) 2004;43(11):1346–1352. doi: 10.1093/rheumatology/keh347.
    1. Nukui T, Ehama R, Sakaguchi M, Sonegawa H, Katagiri C, Hibino T, Huh NH. S100A8/A9, a key mediator for positive feedback growth stimulation of normal human keratinocytes. J Cell Biochem. 2008;104(2):453–464. doi: 10.1002/jcb.21639.
    1. Wu F, Zikusoka M, Trindade A, Dassopoulos T, Harris ML, Bayless TM, Brant SR, Chakravarti S, Kwon JH. MicroRNAs are differentially expressed in ulcerative colitis and alter expression of macrophage inflammatory peptide-2 alpha. Gastroenterology. 2008;135(5):1624–1635. doi: 10.1053/j.gastro.2008.07.068. e1624.
    1. Fits L Van Der, Kant M, Wel LI Van Der, Prens EP. Polymorphisms in the interferon regulatory factor-1 promoter are not associated with psoriasis and do not influence IFN-alpha-induced Th1 polarization. J Interferon Cytokine Res. 2007;27(10):841–846. doi: 10.1089/jir.2007.0013.
    1. Bucht A, Larsson P, Weisbrot L, Thorne C, Pisa P, Smedegard G, Keystone EC, Gronberg A. Expression of interferon-gamma (IFN-gamma), IL-10, IL-12 and transforming growth factor-beta (TGF-beta) mRNA in synovial fluid cells from patients in the early and late phases of rheumatoid arthritis (RA) Clin Exp Immunol. 1996;103(3):357–367.
    1. Gasche C, Bakos S, Dejaco C, Tillinger W, Zakeri S, Reinisch W. IL-10 secretion and sensitivity in normal human intestine and inflammatory bowel disease. J Clin Immunol. 2000;20(5):362–370. doi: 10.1023/A:1006672114184.
    1. Moschen AR, Kaser A, Enrich B, Mosheimer B, Theurl M, Niederegger H, Tilg H. Visfatin, an adipocytokine with proinflammatory and immunomodulating properties. J Immunol. 2007;178(3):1748–1758.
    1. Nowell MA, Richards PJ, Fielding CA, Ognjanovic S, Topley N, Williams AS, Bryant-Greenwood G, Jones SA. Regulation of pre-B cell colony-enhancing factor by STAT-3-dependent interleukin-6 trans-signaling: implications in the pathogenesis of rheumatoid arthritis. Arthritis Rheum. 2006;54(7):2084–2095. doi: 10.1002/art.21942.
    1. Becker KG. Comparative genetics of type 1 diabetes and autoimmune disease: common loci, common pathways? Diabetes. 1999;48(7):1353–1358. doi: 10.2337/diabetes.48.7.1353.

Source: PubMed

Sponsors en medewerkers

Medische omstandigheden

Geneesmiddelinterventies

3
Abonneren