into subtypes. Further studies are required for this purpose.

22 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

**Author details**

**References**

318-24.

Junichi Tani and Yuji Hiromatsu

School of Medicine, Kurume, Fukuoka, Japan

roid. (2004). , 14(12), 1047-55.

roid (2000). , 10, 99-100.

ty. Immunol Res. (2012). Mar 29 in press.

[21] Ban, Y, Davies, T. F, Greenberg, D. A, Concepcion, E. S, Osman, R, Oashi, T, & To‐ mer, Y. Arginine at position 74 of the HLA-DR beta1 chain is associated with Graves' disease. Genes Immun. (2004). , 5, 203-208.

asthma and the level of serum total IgE. Am J Respir Crit Care Med. 20076; , 175,

Genetic Susceptibility to Graves' Ophthalmopathy

http://dx.doi.org/10.5772/53441

25

[31] Jacobson, E. M, Huber, A. K, Akeno, N, Sivak, M, Li, C. W, Concepcion, E, Ho, K, & Tomer, Y. A CD40 Kozak sequence polymorphism and susceptibility to antibodyme‐ diated autoimmune conditions: the role of CD40 tissuespecific expression. Genes Im‐

[32] Cloutier, J. F, & Veillette, A. Cooperative inhibition of T-cell antigen receptor signal‐ ing by a complex between a kinase and a phosphatase. J Exp Med. (1999). , 189,

[33] Ichimura, M, Kaku, H, Fukutani, T, Koga, H, Mukai, T, Miyake, I, Yamada, K, Koda, Y, & Hiromatsu, Y. Associations of protein tyrosine phosphatase nonreceptor 22 (PTPN22) gene polymorphisms with susceptibility to Graves' disease in a Japanese

[34] Vang, T, Congia, M, Macis, M. D, Musumeci, L, Orru, V, Zavattari, P, Nika, K, Tautz, L, Tasken, K, Cucca, F, Mustelin, T, & Bottini, N. Autoimmune-associated lymphoid tyrosine phosphatase is a gain-of-function variant. Nat Genet. (2005). , 37, 1317-1319.

[35] Bottini, N, Musumeci, L, Alonso, A, Rahmouni, S, Nika, K, & Rostamkhani, M. Mac‐ Murray J, Meloni GF, Lucarelli P, Pellecchia M, Eisenbarth GS, Comings D, Mustelin T. A functional variant of lymphoid tyrosine phosphatase is associated with type I

[36] Josefowicz, S. Z, Lu, L. F, & Rudensky, A. Y. Regulatory T cells: mechanisms of dif‐

[37] Gangi, E, Vasu, C, Cheatem, D, & Prabhakar, B. S. I. L-10-p. r. o. d. u. c. i. n. g C. D4+C. D Regulatory T cells play a critical role in granulocyte-macrophage colonystimulating factor induced suppression of experimental autoimmune thyroiditis. J

[38] Saitoh, O, & Nagayama, Y. Regulation of Graves' hyperthyroidism with naturally oc‐ curring CD4+CD25+ regulatory T cells in a mouse model. Endocrinology. (2006). ,

[39] Ban, Y, Tozaki, T, Tobe, T, Ban, Y, Jacobson, E. M, Concepcion, E. S, & Tomer, Y. The regulatory T cell gene FOXP3 and genetic susceptibility to thyroid autoimmunity: an association analysis in Caucasian and Japanese cohorts. J Autoimmun. (2007). , 28,

[40] Czarnocka, B. Thyroperoxidase, thyroglobulin, Na(+)/I(-) symporter, pendrin in thy‐

[41] Tomer, Y, Ban, Y, Concepcion, E, Barbesino, G, Villanueva, R, Greenberg, D. A, & Davies, T. F. Common and unique susceptibility loci in Graves and Hashimoto dis‐

roid autoimmunity. Front Biosci. (2011). , 16, 783-802.

ferentiation and function. Annu Rev Immunol. (2012). , 30, 531-64.

775-782.

111-121.

mun. (2007). , 8, 205-214.

population. Thyroid. (2008). , 18, 625-30.

diabetes. Nat Genet. (2004). , 36, 337-338.

Immunol. (2005). , 174, 7006-7013.

147, 2417-2422.

201-207.

asthma and the level of serum total IgE. Am J Respir Crit Care Med. 20076; , 175, 775-782.

[31] Jacobson, E. M, Huber, A. K, Akeno, N, Sivak, M, Li, C. W, Concepcion, E, Ho, K, & Tomer, Y. A CD40 Kozak sequence polymorphism and susceptibility to antibodyme‐ diated autoimmune conditions: the role of CD40 tissuespecific expression. Genes Im‐ mun. (2007). , 8, 205-214.

[21] Ban, Y, Davies, T. F, Greenberg, D. A, Concepcion, E. S, Osman, R, Oashi, T, & To‐ mer, Y. Arginine at position 74 of the HLA-DR beta1 chain is associated with Graves'

[22] Teft, W. A, Kirchhof, M. G, & Madrenas, J. A molecular perspective of CTLA-4 func‐

[23] Ueda, H, Howson, J. M, Esposito, L, Heward, J, Snook, H, Chamberlain, G, Rainbow, D. B, Hunter, K. M, & Smith, A. N. Di Genova G, Herr MH, Dahlman I, Payne F, Smyth D, Lowe C, Twells RC, Howlett S, Healy B, Nutland S, Rance HE, Everett V, Smink LJ, Lam AC, Cordell HJ, Walker NM, Bordin C, Hulme J, Motzo C, Cucca F, Hess JF, Metzker ML, Rogers J, Gregory S, Allahabadia A, Nithiyananthan R, Tuomi‐ lehto-Wolf E, Tuomilehto J, Bingley P, Gillespie KM, Undlien DE, Ronningen KS, Gu‐ ja C, Ionescu-Tirgoviste C, Savage DA, Maxwell AP, Carson DJ, Patterson CC, Franklyn JA, Clayton DG, Peterson LB, Wicker LS, Todd JA, Gough SC. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Na‐

[24] Bednarczuk, T, Hiromatsu, Y, Fukutani, T, Jazdzewski, K, Miskiewicz, P, Osikowska, M, & Nauman, J. Association of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) gene polymorphism and non-genetic factors with Graves' ophthalmopathy

[25] Vieland, V. J, Huang, Y, Bartlett, C, Davies, T. F, & Tomer, Y. A multilocus model of the genetic architecture of autoimmune thyroid disorder, with clinical implications.

[26] Banchereau, J, Bazan, F, Blanchard, D, Briere, F, Galizzi, J. P, Van Kooten, C, Liu, Y. J, Rousset, F, Saeland, S, & The, C. D. antigen and its ligand. Annu Rev Immunol.

[27] Tomer, Y, Concepcion, E, & Greenberg, D. A. A C/T Single-nucleotide polymorphism in the region of the CD40 gene is associated with Graves' Disease. Thyroid. (2002). ,

[28] Jacobson, E. M, Concepcion, E, Oashi, T, & Tomer, Y. A Graves' disease-associated kozak sequence single-nucleotide polymorphism enhances the efficiency of CD40 gene translation: A case for translational pathophysiology. Endocrinology. (2005). ,

[29] Kurylowicz, A, Kula, D, Ploski, R, Skorka, A, Jurecka- Lubieniecka, B, Zebracka, J, Steinhof-radwanska, K, Hasse- Lazar, K, Hiromatsu, Y, Jarzab, B, & Bednarczuk, T. Association of CD40 gene polymorphism (C-1T) with susceptibility and phenotype

[30] Park, J. H, Chang, H. S, Park, C. S, Jang, A. S, Park, B. L, Rhim, T. Y, Uh, S. T, Kim, Y. H, Chung, I. Y, & Shin, H. D. Association analysis of CD40 polymorphisms with

in European and Japanese populations. Eur J Endocrinol. (2003). , 148, 13-8.

disease. Genes Immun. (2004). , 5, 203-208.

24 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

tion. Annu Rev Immunol. (2006). , 24, 65-97.

ture. (2003). , 423, 506-511.

(1994). , 12, 881-922.

12, 1129-1135.

146, 2684-2691.

Am J Hum Genet. (2008). , 82, 1349-1356.

of Graves' disease. Thyroid. (2005). , 15, 1119-1124.

eases: results of whole-genome screening in a data set of 102 multiplex families. Am J Hum Genet. (2003). , 73, 736-747.

X, Chen, Y, Sheng, Y, Li, R. Y, Su, Q, Gao, L, Jia, W. P, Jin, L, Chen, M. D, Chen, S. J, Chen, Z, & Chen, J. L. Functional SNPs in the SCGB3A2 promoter are associated with

Genetic Susceptibility to Graves' Ophthalmopathy

http://dx.doi.org/10.5772/53441

27

susceptibility to Graves' disease. Hum. Mol. Genet. (2009). , 18, 1156-1170.

bial pathogenesis. Cell Microbiol. (2009). , 11, 1160-1169.

diabetes. Nat. Genet. (2007). , 39, 857-864.

Addison's disease. BMC Med. Genet. (2009).

(2002). , 70, 718-725.

143-147.

(2010). , 42, 70-72.

[52] Areschoug, T, & Gordon, S. Scavenger receptors: role in innate immunity and micro‐

[53] Niimi, T, Munakata, M, Keck-waggoner, C. L, Popescu, N. C, Levitt, R. C, Hisada, M, & Kimura, S. A polymorphism in the human UGRP1 gene promoter that regulates transcription is associated with an increased risk of asthma. Am. J. Hum. Genet.

[54] Todd, J. A, Walker, N. M, Cooper, J. D, Smyth, D. J, Downes, K, Plagnol, V, Bailey, R, Nejentsev, S, Field, S. F, Payne, F, Lowe, C. E, Szeszko, J. S, Hafler, J. P, & Zeitels, L. Yang JHM, Vella A, Nutland S, Stevens HE, Schuilenburg H, Coleman G, Maisuria M, Meadows W, Smink LJ, Healy B, Burren OS, Lam AAC, Ovington NR, Allen J, Adlem E, Leung HT, Wallace C, Howso, JMM, Guja C, Ionescu-Tirgoviste C, Sim‐ monds MJ, Heward JM, Gough SCL, Dunger DB, Wicker LS, Clayton DG. Robust as‐ sociations of four new chromosome regions from genome-wide analyses of type 1

[55] Penna-martinez, M, Ramos-lopez, E, Robbers, I, Kahles, H, Hahner, S, Willenberg, H, Reisch, N, Seidl, C, Segni, M, & Badenhoop, K. The rs1990760 polymorphism within the IFIH1 locus is not associated with Graves' disease, Hashimoto's thyroiditis and

[56] Zhao, Z. F, Cui, B, Chen, H. Y, Wang, S, Li, I, Gu, X. J, Qi, L, Li, X. Y, Ning, G, Zhao, Y. J, & The, A. T polymorphism in the interferon induced helicase gene does not con‐ fer susceptibility to Graves' disease in Chinese population. Endocrine. (2007). , 32,

[57] Ban, Y, Tozaki, T, Taniyama, M, Nakano, Y, Ban, Y, & Hirano, T. Genomic polymor‐ phism in the interferon-induced helicase (IFIH1) gene does not confer susceptibility to autoimmune thyroid disease in the Japanese population. Horm. Metab. Res.

[58] Chistiakov, D. A. Interferon Induced with Helicase C Domain 1 (IFIH1) and Virus-

[59] Kurylowicz, A, Hiromatsu, Y, Jurecka-lubieniecka, B, Kula, D, Kowalska, M, Ichi‐ mura, M, Koga, H, Kaku, H, Bar-andziak, E, Nauman, J, Jarzab, B, Ploski, R, & Bed‐ narczuk, T. Association of NFKB1-94ins/del ATTG promoter polymorphism with susceptibility to and phenotype of Graves' disease. Genes Immun. (2007). , 8, 532-8.

[60] Mclachlan, S. M, & Rapoport, B. Why measure thyroglobulin autoantibodies rather

Induced Autoimmunity: A Review. Viral Immunol. (2010). , 23, 3-15.

than thyroid peroxidase autoantibodies? Thyroid. (2004). , 14, 510-520.

X, Chen, Y, Sheng, Y, Li, R. Y, Su, Q, Gao, L, Jia, W. P, Jin, L, Chen, M. D, Chen, S. J, Chen, Z, & Chen, J. L. Functional SNPs in the SCGB3A2 promoter are associated with susceptibility to Graves' disease. Hum. Mol. Genet. (2009). , 18, 1156-1170.

[52] Areschoug, T, & Gordon, S. Scavenger receptors: role in innate immunity and micro‐ bial pathogenesis. Cell Microbiol. (2009). , 11, 1160-1169.

eases: results of whole-genome screening in a data set of 102 multiplex families. Am J

[42] Hodge, S. E, Ban, Y, Strug, L. J, Greenberg, D. A, Davies, T. F, Concepcion, E. S, Villa‐ nueva, R, & Tomer, Y. Possible interaction between HLA-DRbeta1 and thyroglobulin

[43] Stefan, M, Jacobson, E. M, Huber, A. K, Greenberg, D. A, Li, C. W, Skrabanek, L, Conception, E, Fadlalla, M, Ho, K, & Tomer, Y. A novel variant of the thyroglobulin promoter triggers thyroid autoimmunity through an epigenetic interferon alpha-

[44] Tani, J, Mori, K, Hoshikawa, S, Nakazawa, T, Satoh, J, Nakagawa, Y, Ito, S, & Yoshi‐ da, K. Prevention of lymphocytic thyroiditis in iodide-treated non-obese diabetic mice lacking interferon regulatory factor-1. Eur J Endocrinol. (2002). , 147, 809-14. [45] Brand, O. J, Barrett, J. C, Simmonds, M. J, Newby, P. R, Mccabe, C. J, Bruce, C. K, Ky‐ sela, B, Carr-smith, J. D, Brix, T, Hunt, P. J, Wiersinga, W. M, Hegedus, L, & Connell, J. Wass JAH, Franklyn JA, Weetman AP, Heward JM, Gough SCL. Association of the thyroid stimulating hormone receptor gene (TSHR) with Graves' disease. Hum. Mol.

[46] Chen, C. R, Pichurin, P, Nagayama, Y, Latrofa, F, Rapoport, B, & Mclachlan, S. M. The thyrotropin receptor autoantigen in Graves disease is the culprit as well as the

[47] Wellcome Trust Case Control ConsortiumAustralo-Anglo- American Spondylitis Consortium (TASC). Association scan of 14,500 nonsynonymous SNPs in four diseas‐

[48] Kochi, Y, Yamada, R, Suzuki, A, Harley, J. B, Shirasawa, S, Sawada, T, Bae, S. C, To‐ kuhiro, S, Chang, X, Sekine, A, Takahashi, A, Tsunoda, T, Ohnishi, Y, Kaufman, K. M, Kang, C. P, Kang, C, Otsubo, S, Yumura, W, Mimori, A, Koike, T, Nakamura, Y, Sasazuki, T, & Yamamoto, K. A functional variant in FCRL3, encoding Fc receptorlike 3, is associated with rheumatoid arthritis and several autoimmunities. Nat. Gen‐

[49] Gu, L. Q, Zhu, W, Zhao, S. X, Zhao, L, Zhang, M. J, Cui, B, Song, H. D, Ning, G, & Zhao, Y. J. Clinical associations of the genetic variants of CTLA-4, Tg, TSHR, PTPN22, PTPN12 and FCRL3 in patients with Graves' disease. Clin. Endocrinol.

[50] Swainson, L. A, Mold, J. E, Bajpai, U. D, & Mccune, J. M. Expression of the autoim‐ mune susceptibility gene FcRL3 on human regulatory t cells is associated with dys‐ function and high levels of programmed cell death-1. J. Immunol. (2010). , 184,

[51] Song, H. D, Liang, J, Shi, J. Y, Zhao, S. X, Liu, Z, Zhao, J. J, Peng, Y. D, Gao, G. Q, Tao, J, Pan, C. M, Shao, L, Cheng, F, Wang, Y, Yuan, G. Y, Xu, C, Han, B, Huang, W, Chu,

es identifies autoimmunity variants. Nat. Genet. (2007). , 39, 1329-1337.

variants in Graves' disease. Thyroid. (2006). , 16, 351-355.

modulated mechanism. J. Biol. Chem. (2011). , 286, 31168-79.

Hum Genet. (2003). , 73, 736-747.

26 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

Genet. (2009). , 18, 1704-1713.

et. (2005). , 37, 478-485.

(Oxf). (2010). , 72, 248-55.

3639-3647.

victim. J. Clin. Invest. (2003). , 111, 1897-1904.

[61] Zaletel, K, & Gaberscek, S. Hashimoto's Thyroiditis: From Genes to the Disease. Curr Genom. (2011). , 12, 576-588.

and intractability of autoimmune thyroid disease. Clin. Exp. Immunol. (2008). , 151,

Genetic Susceptibility to Graves' Ophthalmopathy

http://dx.doi.org/10.5772/53441

29

[74] Tani, J, Gopinath, B, Nguyen, B, & Wall, J. R. Extraocular muscle autoimmunity and orbital fat inflammation in thyroid-associated ophthalmopathy. Expert Rev Clin Im‐

[75] Gerding, M N, Van Der Meer, J W, Broenink, M, Bakker, O, Wiersinga, W. M, & Prummel, M. F. Association of thyrotrophin receptor antibodies with the clinical fea‐

tures of Graves' ophthalmopathy. Clin. Endocrinol. (Oxf). (2000). , 52, 267-271. [76] Ponto, K. A, Kanitz, M, Olivo, P. D, Pitz, S, Pfeiffer, N, & Kahaly, G. J. Clinical rele‐ vance of thyroid-stimulating immunoglobulins in Graves' ophthalmopathy.Ophthal‐

[77] Hunt, P. J, Marshall, S. E, Weetman, A. P, Bell, J. I, Wass, J. A, & Welsh, K. I. Cytokine gene polymorphisms in autoimmune thyroid disease. J. Clin. Endocrinol. Metab.

[78] Kumar, S, & Bahn, R. S. Relative overexpression of macrophage derived cytokines in orbital adipose tissue from patients with Graves' ophthalmopathy. J. Clin. Endocri‐

[79] Durrani, O. M, Reuser, T. Q, & Murray, P. I. Infliximab: a novel treatment for sightthreatening thyroid associated ophthalmopathy. Orbit. (2005). , 24, 117-119.

[80] Khalilzadeh, O, Noshad, S, Rashidi, A, & Amirzargar, A. Graves' Ophthalmopathy:

[81] Sims, J. E, Smith, D. E, & The, I. L. family: regulators of immunity. Nat. Rev. Immu‐

[82] Muhlberg, T, Joba, W, Spitzweg, C, Schworm, H. D, Heberling, H. J, & Heufelder, A. E. Interleukin-1 receptor antagonist ribonucleic acid and protein expression by cul‐ tured Graves' and normal orbital fibroblasts is differentially modulated by dexame‐

[83] Tan, G. H, Dutton, C. M, Bahn, R. S, & Interleukin-1, I. L. receptor antagonist and soluble IL-1 receptor inhibit IL-1-induced glycosaminoglycan production in cultured human orbital fibroblasts from patients with Graves' ophthalmopathy. J. Clin. Endo‐

[84] Cuddihy, R. M, & Bahn, R. S. Lack of an association between alleles of interleukin-1 alpha and interleukin-1 receptor antagonist genes and Graves' disease in a North American Caucasian population. J.Clin. Endocrinol. Metab. (1996). , 81, 4476-4478.

thasone and irradiation. J. Clin. Endocrinol. Metab. (2000). , 85, 734-742.

A Review of Immunogenetics. Curr Genom. (2011). , 12, 564-575.

[73] Bahn, R. S. Graves' ophthalmopathy. N. Engl. J. Med. (2010). , 362, 726-738.

379-382.

munol. (2007). , 3, 299-311.

mology. (2011). , 118, 2279-85.

nol. Metab. (2003). , 88, 4246-4250.

crinol. Metab. (1996). , 81, 449-452.

(2000). , 85, 1984-1988.

nol. (2010). , 10, 89-102.

and intractability of autoimmune thyroid disease. Clin. Exp. Immunol. (2008). , 151, 379-382.

[73] Bahn, R. S. Graves' ophthalmopathy. N. Engl. J. Med. (2010). , 362, 726-738.

[61] Zaletel, K, & Gaberscek, S. Hashimoto's Thyroiditis: From Genes to the Disease. Curr

[62] Tani, J, Yoshida, K, Fukazawa, H, Kiso, Y, Sayama, N, Mori, K, Aizawa, Y, Hori, H, Nakasato, N, & Abe, K. Hyperthyroid Graves' disease and primary hypothyroidism caused by TSH receptor antibodies in monozygotic twins: case reports. Endocri J.

[63] Villanueva, R, Greenberg, D. A, Davies, T. F, & Tomer, Y. Sibling recurrence risk in

[64] Tandon, N, Zhang, L, & Weetman, A. P. HLA associations with Hashimoto's thyroi‐

[65] Kavvoura, F. K, Akamizu, T, Awata, T, Ban, Y, Chistiakov, D. A, Frydecka, I, Gha‐ deri, A, Gough, S. C, Hiromatsu, Y, Ploski, R, Wang, P. W, Ban, Y, Bednarczuk, T, Chistiakova, E. I, Chojm, M, Heward, J. M, Hiratani, H, Juo, S. H, Karabon, L, Ka‐ tayama, S, Kurihara, S, Liu, R. T, Miyake, I, Omrani, G. H, Pawlak, E, Taniyama, M, Tozaki, T, & Ioannidis, J. P. Cytotoxic T lymphocyte associated antigen 4 gene poly‐ morphisms and autoimmune thyroid disease: a meta-analysis. J. Clin. Endocrinol.

[66] Criswell, L. A, Pfeiffer, K. A, Lum, R. F, Gonzales, B, Novitzke, J, Kern, M, Moser, K. L, Begovich, A. B, Carlton, V. E, Li, W, Lee, A. T, Ortmann, W, Behrens, T. W, & Gre‐ gersen, P. K. Analysis of families in the multiple autoimmune disease genetics con‐ sortium (MADGC) collection: the PTPN22 620W allele associates with multiple

[67] Toubi, E, & Shoenfeld, Y. The role of vitamin D in regulating immune responses. Isr.

[68] Stefani, M, Papi, S, Suver, M, Glavas-obrovac, L, & Karner, I. Association of vitamin D receptor gene 3'-variants with Hashimoto's thyroiditis in the Croatian population.

[69] Ban, Y, Greenberg, D. A, Concepcion, E, Skrabanek, L, Villanueva, R, & Tomer, Y. Amino acid substitutions in the thyroglobulin gene are associated with susceptibility to human and murine autoimmune thyroid disease. Proc Natl Acad Sci USA. (2003). ,

[70] Ito, C, Watanabe, M, Okuda, N, Watanabe, C, & Iwatani, Y. Association between the severity of Hashimoto's disease and the functional +874A/T polymorphism in the in‐

[71] Nanba, T, Watanabe, M, Akamizu, T, & Iwatani, Y. The-590CC genotype in the IL4 gene as a strong predictive factor for the development of hypothyroidism in Hashi‐

[72] Yamada, H, Watanabe, M, Nanba, T, Akamizu, T, Iwatani, Y, & The, T. C polymor‐ phism in the transforming growth factor- beta1 gene is associated with the severity

autoimmune phenotypes. Am J Hum Genet. (2005). , 76, 561-571.

autoimmune thyroid disease. Thyroid. (2003). , 13, 761-764.

ditis. Clin. Endocrinol. (Oxf). (1991). , 34, 383-386.

Genom. (2011). , 12, 576-588.

28 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

Metab. (2007). , 92, 3162-3170.

Med. Assoc. J. (2010). , 12, 174-175.

100, 15119-15124.

Int. J. Immunogenet. (2008). , 35, 125-131.

terferon-gamma gene. Endocr. J. (2006). , 53, 473-478.

moto disease. Clin. Chem. (2008). , 54, 621-623.

(1998). , 45, 117-121.

[85] Liu, N, Li, X, Liu, C, Zhao, Y, Cui, B, & Ning, G. The association of interleukin-1alpha and interleukin-1beta polymorphisms with the risk of Graves' disease in a case-con‐ trol study and meta-analysis. Hum. Immunol. (2010). , 71, 397-401.

[96] Chng, C. L, Seah, L. L, & Khoo, D. H. Ethnic differences in the clinical presentation of Graves' ophthalmopathy. Best Pract Res Clin Endocrinol Metab. (2012). , 26, 249-58.

Genetic Susceptibility to Graves' Ophthalmopathy

http://dx.doi.org/10.5772/53441

31

[97] Stan, M. N, & Bahn, R. S. Risk factors for development or deterioration of Graves'

[98] Yin, X, Latif, R, Bahn, R, & Davies, T. F. Genetic Profiling in Graves' Disease: Further Evidence for Lack of a Distinct Genetic Contribution to Graves' Ophthalmopathy.

[99] Werner, S. C. Classification of the eye changes of Graves' disease. American Journal

[100] El-Kaissi, S, Frauman, A. G, & Wall, J. R. Thyroid-associated ophthalmopathy: a prac‐ tical guide to classification, natural history and management. Intern Med J. (2004). ,

[101] El-Kaissi, S, & Wall, J. R. Determinants of extraocular muscle volume in patients with

[102] Hiromatsu, Y, Kojima, K, Ishisaka, N, Tanaka, K, Sato, M, Nonaka, K, Nishimura, H, & Nishida, H. Role of magnetic resonance imaging in thyroid-associated ophthalm‐ opathy: its predictive value for therapeutic outcome of immunosuppressive therapy.

[103] Volpe, N. J, & Sbarbaro, J. A. Gendron Livingston K, Galetta SL, Liu GT, Balcer LJ. Occult thyroid eye disease in patients with unexplained ocular misalignment identi‐ fied by standardized orbital echography. Am J Ophthalmol. (2006). , 142, 75-81.

ophthalmopathy. Thyroid. (2010). , 20, 777-83.

of Ophthalmology. (1969). , 68, 646-648.

Graves' disease. J Thyroid Res. (2012).

Thyroid. (1992). , 2, 299-305.

Thyroid. (2012). , 7, 730-736.

34, 482-91.

[96] Chng, C. L, Seah, L. L, & Khoo, D. H. Ethnic differences in the clinical presentation of Graves' ophthalmopathy. Best Pract Res Clin Endocrinol Metab. (2012). , 26, 249-58.

[85] Liu, N, Li, X, Liu, C, Zhao, Y, Cui, B, & Ning, G. The association of interleukin-1alpha and interleukin-1beta polymorphisms with the risk of Graves' disease in a case-con‐

[86] Fukutani, T, Hiromatsu, Y, Kaku, H, Miyake, I, Mukai, T, Imamura, Y, Kohno, S, Ta‐ kane, N, Shoji, S, Otabe, S, & Yamada, K. A polymorphism of interferon-gamma gene associated with changes of anti-thyrotropin receptor antibodies induced by antithy‐ roid drug treatment for Graves' disease in Japanese patients. Thyroid. (2004). , 14-93.

[87] Anvari, M, Khalilzadeh, O, Esteghamati, A, Esfahani, S. A, Rashidi, A, Etemadi, A, Mahmoudi, M, & Amirzargar, A. A. Genetic susceptibility to Graves' ophthalmop‐ athy: the role of polymorphisms in proinflammatory cytokine genes. Eye (Lond).

[88] Vaidya, B, Imrie, H, Perros, P, Dickinson, J, Mccarthy, M. I, Kendall-taylor, P, & Pearce, S. H. Cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphism con‐ fers susceptibility to thyroid associated orbitopathy. Lancet. (1999). , 354, 743-744.

[89] Bednarczuk, T, Gopinath, B, Ploski, R, & Wall, J. R. Susceptibility genes in Graves' ophthalmopathy: searching for a needle in a haystack? Clin. Endocrinol. (Oxf).

[90] Bednarczuk, T, Hiromatsu, Y, Seki, N, Ploski, R, Fukutani, T, Kurylowicz, A, Jazd‐ zewski, K, Chojnowski, K, Itoh, K, & Nauman, J. Association of tumor necrosis factor and human leukocyte antigen DRB1 alleles with Graves' ophthalmopathy. Hum. Im‐

[91] Lavard, L, Madsen, H. O, Perrild, H, Jacobsen, B. B, & Svejgaard, A. HLA class II as‐ sociations in juvenile Graves' disease: indication of a strong protective role of the

[92] Weetman, A. P, Zhang, L, Webb, S, & Shine, B. Analysis of HLADQB and HLA-DPB alleles in Graves' disease by oligonucleotide probing of enzymatically amplified

[93] Kretowski, A, Wawrusiewicz, N, Mironczuk, K, Mysliwiec, J, Kretowska, M, & Kinal‐ ska, I. Intercellular adhesion molecule 1 gene polymorphisms in Graves' disease. J.

[94] Zhao, L. Q, Wei, R. L, Cheng, J. W, Cai, J. P, & Li, Y. The expression of intercellular adhesion molecule-1 induced by CD40-CD40L ligand signaling in orbital fibroblasts in patients with Graves' ophthalmopathy. Invest Ophthalmol Vis Sci. (2010). , 51,

[95] Skórka, A, Bednarczuk, T, Bar-andziak, E, Nauman, J, & Ploski, R. Lymphoid tyro‐ sine phosphatase (PTPN22/LYP) variant and Graves' disease in a Polish population: association and gene dose-dependent correlation with age of onset. Clin Endocrinol

DRB1\*0701,DQA1\*0201 haplotype. Tissue Antigens. (1997). , 50, 639-641.

DNA. Clin. Endocrinol. (Oxf). (1990). , 33, 65-71.

Clin. Endocrinol. Metab. (2003). , 88, 4945-4949.

trol study and meta-analysis. Hum. Immunol. (2010). , 71, 397-401.

30 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

(2010). , 24, 1058-1063.

(2007). , 67, 3-19.

4652-60.

(Oxf). (2005). , 62, 679-82.

munol. (2004). , 65, 632-639.

**Chapter 2**

**Gene Polymorphisms of Immunoregulatory Cytokines**

Systemic lupus erythematosus (SLE) is a complex multifactorial autoimmune disease charac‐ terized by loss of immune tolerance and defective immune regulatory mechanisms that leads to B cell hyperactivity and the production of pathogenic autoantibodies directed against a wide range of autoantigens, and particularly nuclear antigens [Liossis et al., 1996; Yurasov et al., 2006; Mandik-Nayak et al., 2008]. In common, the complex interaction of genetic, environ‐ mental, as well as immunological factors causes the breaking of self-tolerance of the immune system and leads to development of autoimmunity. The immune system has various mecha‐ nisms at the cellular and molecular level that negatively regulate immune responses and counteract establishment of chronic and destructive immunity [Nakken et al., 2012]. A number of circulating cytokine abnormalities have been reported in SLE and recent advances have revealed new insights in cytokine regulation of autoimmune inflammatory responses [Diveu et al., 2008]. In particular, the production of Interleukin-10 (IL-10) and transforming growth factor-β1 (*TGF*-β1), the two main Treg cytokines that suppress the inflammatory response, has been found to be deeply deregulated in SLE patients, so they have been considered essential

Given the importance of cytokines in immune system regulation, these molecules are of high interest not only in the "effector phase" of autoimmune disease in which self-tolerance has already been broken, but also in the "initiation phase" of autoimmunity, in which a lasting immune response against self antigens is first generated. Recently, it has been suggested that initial susceptibility to autoimmune disease lies at least partly in the genetics of cytokine regulation, and that many genetic polymorphisms affecting cytokine patterns could alter thresholds for immune responses, resulting in pro-inflammatory presentation of self antigens

and reproduction in any medium, provided the original work is properly cited.

© 2013 Manolova et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**IL-10 and TGF-β1 in Systemic Lupus Erythematosus**

Irena Manolova, Mariana Ivanova and

elements in the etiopathology of the disease.

Additional information is available at the end of the chapter

Spaska Stanilova

**1. Introduction**

http://dx.doi.org/10.5772/53799

### **Gene Polymorphisms of Immunoregulatory Cytokines IL-10 and TGF-β1 in Systemic Lupus Erythematosus**

Irena Manolova, Mariana Ivanova and Spaska Stanilova

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53799

#### **1. Introduction**

Systemic lupus erythematosus (SLE) is a complex multifactorial autoimmune disease charac‐ terized by loss of immune tolerance and defective immune regulatory mechanisms that leads to B cell hyperactivity and the production of pathogenic autoantibodies directed against a wide range of autoantigens, and particularly nuclear antigens [Liossis et al., 1996; Yurasov et al., 2006; Mandik-Nayak et al., 2008]. In common, the complex interaction of genetic, environ‐ mental, as well as immunological factors causes the breaking of self-tolerance of the immune system and leads to development of autoimmunity. The immune system has various mecha‐ nisms at the cellular and molecular level that negatively regulate immune responses and counteract establishment of chronic and destructive immunity [Nakken et al., 2012]. A number of circulating cytokine abnormalities have been reported in SLE and recent advances have revealed new insights in cytokine regulation of autoimmune inflammatory responses [Diveu et al., 2008]. In particular, the production of Interleukin-10 (IL-10) and transforming growth factor-β1 (*TGF*-β1), the two main Treg cytokines that suppress the inflammatory response, has been found to be deeply deregulated in SLE patients, so they have been considered essential elements in the etiopathology of the disease.

Given the importance of cytokines in immune system regulation, these molecules are of high interest not only in the "effector phase" of autoimmune disease in which self-tolerance has already been broken, but also in the "initiation phase" of autoimmunity, in which a lasting immune response against self antigens is first generated. Recently, it has been suggested that initial susceptibility to autoimmune disease lies at least partly in the genetics of cytokine regulation, and that many genetic polymorphisms affecting cytokine patterns could alter thresholds for immune responses, resulting in pro-inflammatory presentation of self antigens

© 2013 Manolova et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and the subsequent misdirection of adaptive immunity against self which is observed in autoimmune disease [Kariuki et al., 2010]

2002]. In particular, IL-10 knockout mice were demonstrated to develop severe lupus, with earlier appearance of skin lesions, increased lymphadenopathy, more severe glomeruloneph‐ ritis, and higher mortality than their IL-10-intact littermate controls [Yin et al., 2002]. Interest‐ ingly, the injection of IL-10 could prevent the occurrence of the disease in such models [Yin et al., 2002]. The authors of the study suggested that the contradictory role of IL-10 in lupus could be explained taking into account the phase of the disease. IL-10 deficiency enhanced IFN-γ production in the CD4 and CD8 lineages, and that, in turn, was associated with increased production of IgG2a anti-dsDNA antibodies, especially at the early stages of disease develop‐ ment. In contrast, at later phases of disease, excessive amounts of IL-10 production led to enhanced autoantibody production and subsequent formation of pathogenic autoantibody– antigen complexes [Yin et al., 2002]. However, Llorente and colleagues demonstrated that constitutive IL-10 production by monocytes and B cells in healthy members of multicase families with SLE was significantly higher than that of healthy unrelated controls, but was similar to that of SLE patients, thus suggesting that a genetically controlled high innate IL-10

Gene Polymorphisms of Immunoregulatory Cytokines IL-10 and TGF-β1 in Systemic Lupus Erythematosus

http://dx.doi.org/10.5772/53799

35

Recently, experimental studies have demonstrated an association between *TGF*-β1 and the development of autoimmunity [Aoki et al., 2005]. TGF-β1 belongs to a large family of multi‐ functional proteins, secreted by a variety of cell types that act as signal molecules in controlling a great number of biological processes. Like IL-10, it is a highly pleiotropic cytokine with an important role in maintaining immune homeostasis [Li et al., 2006]. There are three TGF-β isoforms in mammalian cells, TGF-β1, 2 and 3, which use similar signaling pathways and exert overlapping, albeit not identical biological functions. TGF-β1 is involved in many critical cellular processes, including cell growth, extracellular matrix formation, cell motility, hema‐ topoiesis, apoptosis and immune function (Moustakas et al., 2002; Schuster & Krieglstein, 2002). The cells of immune system, including B, T and dendritic cells as well as macrophages, mostly produce TGF-β1, an isoform that is also found in large amounts in the plasma [Flanders and Roberts, 2001]. TGF-β1 has pronounced anti-inflammatory and immunosuppressive functions, the latter being realized by controlling the activation, proliferation, differentiation and survival of all effector immune cells [Rubtsov et al., 2007; Wahl, 1992]. Importantly, TGFβ1 inhibits maturation of dendritic cells and modulates the functions of antigen-presenting cells, reducing the macrophage production of IL-1, IL-6, and TNF-α [Fainaru et al., 2007]. However, immunosuppressive effect of TGF-β1 was most pronounced for T cells [Rubtsov et al., 2007; Li et al., 2006]. It inhibits T cell proliferation and production of IL-2, differentiation of Th1, Th2, and cytotoxic T lymphocytes (CTL). TGF-β1 suppresses IFN-γ production from Th1, NK cells and CTL, as well as their cytotoxic activity. TGF-β1 plays an essential role in the functioning and survival of regulatory T cells (Treg), and *de novo* generation of Foxp3+ Treg

T lymphocytes in the periphery [Pyzik et al., 2007; Selvaraj et al.,

2007; Zheng et al., 2007]. Furthermore, recent study reveals a role for TGF-β1 as effector molecule of Foxp3+ Treg cells [Li et al., 2007]. In contrast to IL-10, TGF-β1 is also an important negative regulator of B cell differentiation and proliferation, inhibiting the production of most

immunoglobulin isotypes except IgA [Lebman and Edmiston, 1999].

production may predispose to SLE development. [Llorente et al.,1997]

**2.2.** *TGF***-β1**

from naive CD4+ CD25-

Genetic polymorphisms have emerged in recent years as important determinants of disease susceptibility and severity. Polymorphisms are naturally occurring DNA sequence variations, which differ from gene mutations in that they occur in the normal healthy population and have a frequency of at least 1%. Approximately 90% of DNA polymorphisms are single nucleotide polymorphisms (SNPs) due to single base substitutions. Others include insertion/deletion polymorphisms, minisatellite and microsatellite polymorphisms. Although most polymor‐ phisms are functionally neutral, some have effects on regulation of gene expression or on the function of the coded protein. These functional polymorphisms, despite being of low pene‐ trance, could contribute to the differences between individuals in susceptibility to and severity of disease. Many studies have examined the relationship between certain cytokine gene polymorphism, cytokine gene expression in vitro, and the susceptibility to and clinical severity of diseases [Bidwell et al., 1999; Hollegaard and Bidwell, 2006]. Some genetic polymorphisms at the promoter regions of IL-10 and TGF-β1 genes have been associated with cytokine production. Given that the production of these molecules is controlled at genetic level, functional polymorphisms in their promoters could influence the development and severity of the disease. In the present review, we summarize the information about involvement of IL-10 and TGF-β1 genetic variants on SLE appearance and clinical presentation.

#### **2. Role of IL-10 and** *TGF***-β1 in immune regulation and autoimmunity**

#### **2.1. IL-10**

IL-10 is a pleiotropic cytokine with important immunoregulatory functions, which can be produced by both leukocytes and structural cells within tissues, being produced in particular by Tregs *in vivo* [Wakkach et al., 2008]. It is pivotal in inhibiting inflammation and suppresses Th1-mediated immune response through down regulation of proinflmama‐ tory cytokine secretion from both Th1 and activated macrophages (De Waal Malefyt et al., 1993; Fiorentino et al., 1998]. It also inhibits antigen presenting cells by downregulating major histocompatibility complex class II (MHC-II) and B7 expression [Ding et al., 1993]. In addition to these inhibitory actions, IL-10 promotes B-cell-mediated functions, enhanc‐ ing survival, proliferation, differentiation, and antibody production [Rousset et al., 1992]. Hence, increased production of IL-10 could thus explain B cell hyperactivity and autoanti‐ body production, two main features of the immune dysregulation in SLE. In fact, high serum levels of IL-10 have been reported in patients with SLE and they correlated positively with the disease activity [Park et al., 1998].

The role of IL-10 in the pathogenesis of lupus remains controversial. The abnormally elevated amounts of IL-10 detected in serum of patients with SLE [Houssiau et al.,1995] and the observation that anti-IL-10 therapy can down-modulate disease in such patients [Llorente and Riehaud-Patin, 2003] suggest that this cytokine may promote disease. In contrast, mouse models have suggested a preventive role for IL-10 in the pathogenesis of lupus [Yin et al., 2002]. In particular, IL-10 knockout mice were demonstrated to develop severe lupus, with earlier appearance of skin lesions, increased lymphadenopathy, more severe glomeruloneph‐ ritis, and higher mortality than their IL-10-intact littermate controls [Yin et al., 2002]. Interest‐ ingly, the injection of IL-10 could prevent the occurrence of the disease in such models [Yin et al., 2002]. The authors of the study suggested that the contradictory role of IL-10 in lupus could be explained taking into account the phase of the disease. IL-10 deficiency enhanced IFN-γ production in the CD4 and CD8 lineages, and that, in turn, was associated with increased production of IgG2a anti-dsDNA antibodies, especially at the early stages of disease develop‐ ment. In contrast, at later phases of disease, excessive amounts of IL-10 production led to enhanced autoantibody production and subsequent formation of pathogenic autoantibody– antigen complexes [Yin et al., 2002]. However, Llorente and colleagues demonstrated that constitutive IL-10 production by monocytes and B cells in healthy members of multicase families with SLE was significantly higher than that of healthy unrelated controls, but was similar to that of SLE patients, thus suggesting that a genetically controlled high innate IL-10 production may predispose to SLE development. [Llorente et al.,1997]

#### **2.2.** *TGF***-β1**

and the subsequent misdirection of adaptive immunity against self which is observed in

Genetic polymorphisms have emerged in recent years as important determinants of disease susceptibility and severity. Polymorphisms are naturally occurring DNA sequence variations, which differ from gene mutations in that they occur in the normal healthy population and have a frequency of at least 1%. Approximately 90% of DNA polymorphisms are single nucleotide polymorphisms (SNPs) due to single base substitutions. Others include insertion/deletion polymorphisms, minisatellite and microsatellite polymorphisms. Although most polymor‐ phisms are functionally neutral, some have effects on regulation of gene expression or on the function of the coded protein. These functional polymorphisms, despite being of low pene‐ trance, could contribute to the differences between individuals in susceptibility to and severity of disease. Many studies have examined the relationship between certain cytokine gene polymorphism, cytokine gene expression in vitro, and the susceptibility to and clinical severity of diseases [Bidwell et al., 1999; Hollegaard and Bidwell, 2006]. Some genetic polymorphisms at the promoter regions of IL-10 and TGF-β1 genes have been associated with cytokine production. Given that the production of these molecules is controlled at genetic level, functional polymorphisms in their promoters could influence the development and severity of the disease. In the present review, we summarize the information about involvement of

IL-10 and TGF-β1 genetic variants on SLE appearance and clinical presentation.

positively with the disease activity [Park et al., 1998].

**2. Role of IL-10 and** *TGF***-β1 in immune regulation and autoimmunity**

IL-10 is a pleiotropic cytokine with important immunoregulatory functions, which can be produced by both leukocytes and structural cells within tissues, being produced in particular by Tregs *in vivo* [Wakkach et al., 2008]. It is pivotal in inhibiting inflammation and suppresses Th1-mediated immune response through down regulation of proinflmama‐ tory cytokine secretion from both Th1 and activated macrophages (De Waal Malefyt et al., 1993; Fiorentino et al., 1998]. It also inhibits antigen presenting cells by downregulating major histocompatibility complex class II (MHC-II) and B7 expression [Ding et al., 1993]. In addition to these inhibitory actions, IL-10 promotes B-cell-mediated functions, enhanc‐ ing survival, proliferation, differentiation, and antibody production [Rousset et al., 1992]. Hence, increased production of IL-10 could thus explain B cell hyperactivity and autoanti‐ body production, two main features of the immune dysregulation in SLE. In fact, high serum levels of IL-10 have been reported in patients with SLE and they correlated

The role of IL-10 in the pathogenesis of lupus remains controversial. The abnormally elevated amounts of IL-10 detected in serum of patients with SLE [Houssiau et al.,1995] and the observation that anti-IL-10 therapy can down-modulate disease in such patients [Llorente and Riehaud-Patin, 2003] suggest that this cytokine may promote disease. In contrast, mouse models have suggested a preventive role for IL-10 in the pathogenesis of lupus [Yin et al.,

autoimmune disease [Kariuki et al., 2010]

34 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

**2.1. IL-10**

Recently, experimental studies have demonstrated an association between *TGF*-β1 and the development of autoimmunity [Aoki et al., 2005]. TGF-β1 belongs to a large family of multi‐ functional proteins, secreted by a variety of cell types that act as signal molecules in controlling a great number of biological processes. Like IL-10, it is a highly pleiotropic cytokine with an important role in maintaining immune homeostasis [Li et al., 2006]. There are three TGF-β isoforms in mammalian cells, TGF-β1, 2 and 3, which use similar signaling pathways and exert overlapping, albeit not identical biological functions. TGF-β1 is involved in many critical cellular processes, including cell growth, extracellular matrix formation, cell motility, hema‐ topoiesis, apoptosis and immune function (Moustakas et al., 2002; Schuster & Krieglstein, 2002). The cells of immune system, including B, T and dendritic cells as well as macrophages, mostly produce TGF-β1, an isoform that is also found in large amounts in the plasma [Flanders and Roberts, 2001]. TGF-β1 has pronounced anti-inflammatory and immunosuppressive functions, the latter being realized by controlling the activation, proliferation, differentiation and survival of all effector immune cells [Rubtsov et al., 2007; Wahl, 1992]. Importantly, TGFβ1 inhibits maturation of dendritic cells and modulates the functions of antigen-presenting cells, reducing the macrophage production of IL-1, IL-6, and TNF-α [Fainaru et al., 2007]. However, immunosuppressive effect of TGF-β1 was most pronounced for T cells [Rubtsov et al., 2007; Li et al., 2006]. It inhibits T cell proliferation and production of IL-2, differentiation of Th1, Th2, and cytotoxic T lymphocytes (CTL). TGF-β1 suppresses IFN-γ production from Th1, NK cells and CTL, as well as their cytotoxic activity. TGF-β1 plays an essential role in the functioning and survival of regulatory T cells (Treg), and *de novo* generation of Foxp3+ Treg from naive CD4+ CD25- T lymphocytes in the periphery [Pyzik et al., 2007; Selvaraj et al., 2007; Zheng et al., 2007]. Furthermore, recent study reveals a role for TGF-β1 as effector molecule of Foxp3+ Treg cells [Li et al., 2007]. In contrast to IL-10, TGF-β1 is also an important negative regulator of B cell differentiation and proliferation, inhibiting the production of most immunoglobulin isotypes except IgA [Lebman and Edmiston, 1999].

There are strong evidences to suggest the great importance of this cytokine in the control of autoimmunity [Aoki et al., 2005]. An association between *TGF*-β1 and the development of autoimmunity is clearly demonstrated in studies with complete knockout of *TGF*-β1 in mice or genetic manipulation of its receptors in T cells. TGF-β1 knockout mice and those with impaired TGF-β1 signaling in T cells develop an autoimmune syndrome with multiple organ involvement and death [Shull et al., 1992; Dang et al., 1995; Gorelik et al., 2000; Marie et al., 2006;]. This syndrome resembles SLE and Sjogren's syndrome in humans [Dang et al., 1995] and is characterized by multifocal inflammatory process affecting the heart, brain, lungs, skeletal muscle, liver, stomach, pancreas, salivary glands and other organs, lymfoproliferation, spontaneous activation of autoreactive T lymphocytes and production of autoantibodies [Dang et al., 1995; Shull et al., 1992].

Gene Polymorphisms of Immunoregulatory Cytokines IL-10 and TGF-β1 in Systemic Lupus Erythematosus

http://dx.doi.org/10.5772/53799

37

SLE was recently observed (Lin et al., 2012).

**3.2. TGF-β1 genetic polymorphisms and plasma concentrations of TGF-β1**

SNPs in white populations (Cambien et al.,1996; Grainger at al. 1999).

The TGF-β1 gene is located on chromosome 19 (q13.1-13.3) and several SNPs were described so far in promoter region, in the non-translated region (introns), in the coding region (exons), and in the 3'-UTR region of the gene. They include three polymorphisms in the promoter region (-988C/A, -800G/A, and -509C/T), two polymorphisms located in exon 1 +869T/C (codon 10) and +915G/C (codon 25), one on exon 5 +11929C/T (codon 263), and one in 3'-UTR region of the gene at position +72 [Cambien et al., 1996]. Certain inherited variants in the promoter region of the TGF-β gene (-800G/A and -509C/T) have been associated with higher cytokine circulating concentrations. The -800G/A SNP is located in a consensus cyclic AMP response element binding protein (CREB) half site and may cause reduced affinity for CREB transcription factors whose binding is important for transcription control [Grainger at al. 1999]. The -509C/T is located within a YY1 consen‐ sus binding site and -509T allele has been associated with increased TGF-β1 plasma level [Grainger at al. 1999] and reduced T-cell proliferation [Meng et al., 2005] and a study of twins estimated that the -509C/T polymorphism explained approximately 8% of the genetic variation in TGF-β1 plasma levels (Grainger at al.,1999). Two SNPs, the +868T/C SNP, and the +915G/C SNP, give rise to amino acid substitutions at positions 10 (Leu10Pro) and 25 (Arg25Pro) in the signal peptide of TGF-β1, respectively (Cambien et al.,1996; Awad et al. 1998). The +868T/C SNP was reported to influence steady-state concentrations of *TGFB1* mRNA in peripheral blood mononuclear cells and serum levels of TGF-β1, and the +915G/ C SNP was found to be related to TGF-β1 production in peripheral blood leukocytes (Awad et al., 1998). Another nonsynonymous SNP of *TGFB1*, the +11929C/T SNP (Thr263Ile) is located in exon 5 (Cambien et al.,1996; Awad et al.1998). The 11929C/T SNP is located closely to the site where the latency-associated peptide is cleaved from the active part of the protein (Dubois et al., 1995) and therefore, this SNP may be related to the activation process of TGF-β1, as suggested previously (Cambien et al. 1996). A high degree of linkage disequilibrium was observed between pairs of the −509C/T, 868T/C, 913G/C, and 11929C/T

#### **3. Functional IL-10 and TGF-β1 genetic polymorphisms**

#### **3.1. IL-10 genetic polymorphisms and IL-10 production**

Human IL-10 is secreted mostly by antigen-presenting cells and Treg lymphocytes subset in responsetoseveralactivationstimuli.This cytokinecouldbealsoconstitutivelyproducedatlow levels by immune cells, mainly monocytes, macrophages and dendritic cells. IL-10 is a noncovalent homodimer of 36 kDa with two polypeptide chains and its gene, with GeneBank accession number: X78437, is located on chromosome 1 at 1q31- q32 and a number of polymor‐ phisms in the promoter region have been characterized. In contrast to many other cytokines, the synthesis of IL-10 is regulated by the transcription factors Sp1 and Sp3, which are constitutive‐ lyexpressedbydifferent celltypes [Mooreetal.2001].Ithasbeenrecentlyshownthat c-Junbinds to a highly conserved noncoding sequences (CNS-3) in the IL10 locus, enhancing the expres‐ sion of IL-10, and AP-1 signaling pathway particularly through c-jun transcription and activity strongly affects IL-10 expression in the Th2 cells and monocytes [Wang Z et al., 2005; Dobreva et al., 2009]. Large interindividual differences in the IL-10 inducibility have been observed, which has shown to have a genetic component of over 70%. The IL-10 gene comprises 5 exons, and to date,atleast 49IL10–associatedpolymorphismshavebeenreported,andanevenlargernumber of polymorphisms are recorded in SNP databases (Ensembl Genome Browser, 2006). Promoter polymorphisms have been subject to the most studies, particularly with regard to possible influences on gene transcription and protein production. Three SNPs at -1082(A/G), -819(C/T), -592(C/A) upstream from the transcription start site [D'Alfonso et al., 1995; Turner et al., 1997] have been described as well as additional two microsatellite (CA)n repeats, termed IL10.G (-1.1Kb)andIL10.R(-4Kb)andlocatedat-1151and-3978respectively(EskdaleandGalager,1995; Eskdale et al., 1997).

A complete linkage disequilibrium exists between the alleles present at positions -1082, -819 and -592; so these polymorphisms occurred in tandem These SNPs have been associated with variability in IL-10 production [Eskdale et al., 1998; Turner et al., 1998]. In particular, SNP at position -1082A/G of IL-10 gene has been associated with IL-10 produc‐ tion alone or in haplotypes with other distal SNPs. Turner et al.,1997 have shown that

#### **3.2. TGF-β1 genetic polymorphisms and plasma concentrations of TGF-β1**

There are strong evidences to suggest the great importance of this cytokine in the control of autoimmunity [Aoki et al., 2005]. An association between *TGF*-β1 and the development of autoimmunity is clearly demonstrated in studies with complete knockout of *TGF*-β1 in mice or genetic manipulation of its receptors in T cells. TGF-β1 knockout mice and those with impaired TGF-β1 signaling in T cells develop an autoimmune syndrome with multiple organ involvement and death [Shull et al., 1992; Dang et al., 1995; Gorelik et al., 2000; Marie et al., 2006;]. This syndrome resembles SLE and Sjogren's syndrome in humans [Dang et al., 1995] and is characterized by multifocal inflammatory process affecting the heart, brain, lungs, skeletal muscle, liver, stomach, pancreas, salivary glands and other organs, lymfoproliferation, spontaneous activation of autoreactive T lymphocytes and production of autoantibodies

Human IL-10 is secreted mostly by antigen-presenting cells and Treg lymphocytes subset in responsetoseveralactivationstimuli.This cytokinecouldbealsoconstitutivelyproducedatlow levels by immune cells, mainly monocytes, macrophages and dendritic cells. IL-10 is a noncovalent homodimer of 36 kDa with two polypeptide chains and its gene, with GeneBank accession number: X78437, is located on chromosome 1 at 1q31- q32 and a number of polymor‐ phisms in the promoter region have been characterized. In contrast to many other cytokines, the synthesis of IL-10 is regulated by the transcription factors Sp1 and Sp3, which are constitutive‐ lyexpressedbydifferent celltypes [Mooreetal.2001].Ithasbeenrecentlyshownthat c-Junbinds to a highly conserved noncoding sequences (CNS-3) in the IL10 locus, enhancing the expres‐ sion of IL-10, and AP-1 signaling pathway particularly through c-jun transcription and activity strongly affects IL-10 expression in the Th2 cells and monocytes [Wang Z et al., 2005; Dobreva et al., 2009]. Large interindividual differences in the IL-10 inducibility have been observed, which has shown to have a genetic component of over 70%. The IL-10 gene comprises 5 exons, and to date,atleast 49IL10–associatedpolymorphismshavebeenreported,andanevenlargernumber of polymorphisms are recorded in SNP databases (Ensembl Genome Browser, 2006). Promoter polymorphisms have been subject to the most studies, particularly with regard to possible influences on gene transcription and protein production. Three SNPs at -1082(A/G), -819(C/T), -592(C/A) upstream from the transcription start site [D'Alfonso et al., 1995; Turner et al., 1997] have been described as well as additional two microsatellite (CA)n repeats, termed IL10.G (-1.1Kb)andIL10.R(-4Kb)andlocatedat-1151and-3978respectively(EskdaleandGalager,1995;

A complete linkage disequilibrium exists between the alleles present at positions -1082, -819 and -592; so these polymorphisms occurred in tandem These SNPs have been associated with variability in IL-10 production [Eskdale et al., 1998; Turner et al., 1998]. In particular, SNP at position -1082A/G of IL-10 gene has been associated with IL-10 produc‐ tion alone or in haplotypes with other distal SNPs. Turner et al.,1997 have shown that

[Dang et al., 1995; Shull et al., 1992].

Eskdale et al., 1997).

**3. Functional IL-10 and TGF-β1 genetic polymorphisms**

**3.1. IL-10 genetic polymorphisms and IL-10 production**

36 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

The TGF-β1 gene is located on chromosome 19 (q13.1-13.3) and several SNPs were described so far in promoter region, in the non-translated region (introns), in the coding region (exons), and in the 3'-UTR region of the gene. They include three polymorphisms in the promoter region (-988C/A, -800G/A, and -509C/T), two polymorphisms located in exon 1 +869T/C (codon 10) and +915G/C (codon 25), one on exon 5 +11929C/T (codon 263), and one in 3'-UTR region of the gene at position +72 [Cambien et al., 1996]. Certain inherited variants in the promoter region of the TGF-β gene (-800G/A and -509C/T) have been associated with higher cytokine circulating concentrations. The -800G/A SNP is located in a consensus cyclic AMP response element binding protein (CREB) half site and may cause reduced affinity for CREB transcription factors whose binding is important for transcription control [Grainger at al. 1999]. The -509C/T is located within a YY1 consen‐ sus binding site and -509T allele has been associated with increased TGF-β1 plasma level [Grainger at al. 1999] and reduced T-cell proliferation [Meng et al., 2005] and a study of twins estimated that the -509C/T polymorphism explained approximately 8% of the genetic variation in TGF-β1 plasma levels (Grainger at al.,1999). Two SNPs, the +868T/C SNP, and the +915G/C SNP, give rise to amino acid substitutions at positions 10 (Leu10Pro) and 25 (Arg25Pro) in the signal peptide of TGF-β1, respectively (Cambien et al.,1996; Awad et al. 1998). The +868T/C SNP was reported to influence steady-state concentrations of *TGFB1* mRNA in peripheral blood mononuclear cells and serum levels of TGF-β1, and the +915G/ C SNP was found to be related to TGF-β1 production in peripheral blood leukocytes (Awad et al., 1998). Another nonsynonymous SNP of *TGFB1*, the +11929C/T SNP (Thr263Ile) is located in exon 5 (Cambien et al.,1996; Awad et al.1998). The 11929C/T SNP is located closely to the site where the latency-associated peptide is cleaved from the active part of the protein (Dubois et al., 1995) and therefore, this SNP may be related to the activation process of TGF-β1, as suggested previously (Cambien et al. 1996). A high degree of linkage disequilibrium was observed between pairs of the −509C/T, 868T/C, 913G/C, and 11929C/T SNPs in white populations (Cambien et al.,1996; Grainger at al. 1999).

Although immunosuppressive effects of TGF-β1 have been well established, few studies have investigated serum TGF-β1 levels in autoimmune disorders. In fact, patient with SLE have reduced TGF-β1 production by their peripheral blood lymphocytes (Oshtuka et al., 1998). Hence, reduced TGF-β1 production by immune cells might contribute to the characteristic T cell disregulation, aberrant stimulation of autoreactive B cell, and autoantibody production in SLE patients. Also, it has been reported that decreased serum levels of TGF-β1 in patients with systemic lupus are the most pronounced and constant abnormality in the cytokine levels in these patients [Becker-Merok et al., 2010]. In attempt to elucidate the importance of TGF-β1 for the development of SLE we measured serum levels of TGF-β1 in 53 patients with SLE recruited from 'St Ivan Rilski' University Hospital, Sofia and in 66 healthy controls [unpub‐ lished data]. Serum samples were routinely collected and stored frozen at -20 C until assayed. At the time of sampling, neither of the patients and control subjects had clinical signs or symptoms of intercurrent illness. The concentrations of activated TGF-β1 protein in the serum samples of patients and controls were measured by quantitative sandwich ELISA technique, using commercially available kits (Qantakine®, R&D systems, Abingdon, UK). Before assay, the latent TGF-β1 contained in sera was activated to the immunoreactive form using acid activation and neutralization. The results were calculated by reference to the standard curve and expressed as ng/ml. Table 1 presents the serum concentrations of TGF-β1 in patients and healthy controls.

variation in serum TGF-β1 levels among healthy subjects. This variation might be partly due to the possible influence of some endogenous factors, such as *gender* and age on the production of *TGF*-β1. Given this supposition, we examined *TGF*-β1 levels in the context of age and gender in both healthy and affected individuals. The highest levels of *TGF*-β1 were found in men among the healthy subjects. In SLE patients such a correlation between serum levels of *TGF*β1 and gender was not observed (Table 1), which could be explained with fact, that only men individuals with genetic predisposition to lower *TGF*-β1 production developed SLE or down regulation of *TGF*-β1 by disease progress. Additionally, age was positively correlated with serum *TGF*-β1 in healthy controls. *TGF*-β1 serum levels were lower in individuals under 45 years and higher in those aged over 45 years (P<0.001) among healthy controls as shown in Figure 1. In SLE patients, there was not a relationship between age and serum *TGF*-β1 levels. Thus, healthy individuals showed a pattern in which serum *TGF*-β1 was higher in men and elder people. It seems likely that age- or gender-specific cytokine differences could play a role in the observed age- and gender-related incidence patterns observed in SLE [Petri M, 2002].These data allow us to hypothesis that high levels of serum TGF-β1 may protect against autoimmunity in men as well as low levels of serum TGF-β1 may predispose to the onset of

Gene Polymorphisms of Immunoregulatory Cytokines IL-10 and TGF-β1 in Systemic Lupus Erythematosus

**controls SLE**

**Figure 1.** Mean (±SD) serum *TGF*-β1 concentrations (pg/ml) in SLE patients and healthy controls according to the age

Nowadays, it is considered that the control of TGF-β1 production is complex, but it has been estimated that 54% of its production is under genetic control [Grainger et al., 1999]. In this regard, we analyzed the serum levels of TGF-β1 in relation to various genotypes of -509C/T polymorphism of TGFB1 in 52 healthy controls (n = 21 for CC, 15 for CT, and16 for TT) and in 48 SLE patients (n = 17 for CC, 24 for CT, and 7 for TT). As the mean TGF-β1 levels varied significantly by case-control status (P < 0.001), patients and controls were proceeded separetly for the genotype-serum levels analysis. The results are presented on Figure 2. Among healthy individuals the highest TGF-β1 concentration was detected in individuals with TT genotype (mean ± SD, 56.9 ± 28.6 ng/ml) compared to those with CC genotype (mean ± SD, 29.5 ± 21.1

<45 years >45 yers

http://dx.doi.org/10.5772/53799

39

autoimmunity in younger individuals.

P<0.001

0

of individuals.

20

40

**TGF-beta1 ng/ml**

60

80

100

**Table 1.** *TGF*-β1 concentrations (ng/ml 9) in SLE patients and healthy controls.

Our results showed significantly lower levels of active TGF-β1 in lupus patients compared with healthy individuals (p<0.001). These data are in principal agreement with other studies [Jin et al., 2011; Lu et al., 2004]. In addition to the decreased TGF-β1 serum concentrations in SLE patients, the authors of these studies also reported an association between lower TGF-β1 and disease activity, as well as the development of organ damage [Jin et al., 2011; Lu et al., 2004]. Taken together, these observations support the role of TGF-β1 in SLE pathogenesis and modulation of disease expression. In our study, we also observed a large interindividual variation in serum TGF-β1 levels among healthy subjects. This variation might be partly due to the possible influence of some endogenous factors, such as *gender* and age on the production of *TGF*-β1. Given this supposition, we examined *TGF*-β1 levels in the context of age and gender in both healthy and affected individuals. The highest levels of *TGF*-β1 were found in men among the healthy subjects. In SLE patients such a correlation between serum levels of *TGF*β1 and gender was not observed (Table 1), which could be explained with fact, that only men individuals with genetic predisposition to lower *TGF*-β1 production developed SLE or down regulation of *TGF*-β1 by disease progress. Additionally, age was positively correlated with serum *TGF*-β1 in healthy controls. *TGF*-β1 serum levels were lower in individuals under 45 years and higher in those aged over 45 years (P<0.001) among healthy controls as shown in Figure 1. In SLE patients, there was not a relationship between age and serum *TGF*-β1 levels. Thus, healthy individuals showed a pattern in which serum *TGF*-β1 was higher in men and elder people. It seems likely that age- or gender-specific cytokine differences could play a role in the observed age- and gender-related incidence patterns observed in SLE [Petri M, 2002].These data allow us to hypothesis that high levels of serum TGF-β1 may protect against autoimmunity in men as well as low levels of serum TGF-β1 may predispose to the onset of autoimmunity in younger individuals.

Although immunosuppressive effects of TGF-β1 have been well established, few studies have investigated serum TGF-β1 levels in autoimmune disorders. In fact, patient with SLE have reduced TGF-β1 production by their peripheral blood lymphocytes (Oshtuka et al., 1998). Hence, reduced TGF-β1 production by immune cells might contribute to the characteristic T cell disregulation, aberrant stimulation of autoreactive B cell, and autoantibody production in SLE patients. Also, it has been reported that decreased serum levels of TGF-β1 in patients with systemic lupus are the most pronounced and constant abnormality in the cytokine levels in these patients [Becker-Merok et al., 2010]. In attempt to elucidate the importance of TGF-β1 for the development of SLE we measured serum levels of TGF-β1 in 53 patients with SLE recruited from 'St Ivan Rilski' University Hospital, Sofia and in 66 healthy controls [unpub‐ lished data]. Serum samples were routinely collected and stored frozen at -20 C until assayed. At the time of sampling, neither of the patients and control subjects had clinical signs or symptoms of intercurrent illness. The concentrations of activated TGF-β1 protein in the serum samples of patients and controls were measured by quantitative sandwich ELISA technique, using commercially available kits (Qantakine®, R&D systems, Abingdon, UK). Before assay, the latent TGF-β1 contained in sera was activated to the immunoreactive form using acid activation and neutralization. The results were calculated by reference to the standard curve and expressed as ng/ml. Table 1 presents the serum concentrations of TGF-β1 in patients and

**TGF-β1 concentrations (mean±SD) Significance**

female (n=46) 8.93 ± 3.95 male *vs* female p = 0.82

female (n=53) 26.12 ± 17.34 male *vs* female p <0.001

Our results showed significantly lower levels of active TGF-β1 in lupus patients compared with healthy individuals (p<0.001). These data are in principal agreement with other studies [Jin et al., 2011; Lu et al., 2004]. In addition to the decreased TGF-β1 serum concentrations in SLE patients, the authors of these studies also reported an association between lower TGF-β1 and disease activity, as well as the development of organ damage [Jin et al., 2011; Lu et al., 2004]. Taken together, these observations support the role of TGF-β1 in SLE pathogenesis and modulation of disease expression. In our study, we also observed a large interindividual

healthy controls.

Healthy controls

total (n=53) 8.88 ± 3.79 male (n=7) 8.57 ± 2.72

38 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

total (n=66) 32.99 ± 24.84 male (n=13) 60.98 ± 31.32

Significance SLE *vs* HC p<0.001

**Table 1.** *TGF*-β1 concentrations (ng/ml 9) in SLE patients and healthy controls.

SLE

**Figure 1.** Mean (±SD) serum *TGF*-β1 concentrations (pg/ml) in SLE patients and healthy controls according to the age of individuals.

Nowadays, it is considered that the control of TGF-β1 production is complex, but it has been estimated that 54% of its production is under genetic control [Grainger et al., 1999]. In this regard, we analyzed the serum levels of TGF-β1 in relation to various genotypes of -509C/T polymorphism of TGFB1 in 52 healthy controls (n = 21 for CC, 15 for CT, and16 for TT) and in 48 SLE patients (n = 17 for CC, 24 for CT, and 7 for TT). As the mean TGF-β1 levels varied significantly by case-control status (P < 0.001), patients and controls were proceeded separetly for the genotype-serum levels analysis. The results are presented on Figure 2. Among healthy individuals the highest TGF-β1 concentration was detected in individuals with TT genotype (mean ± SD, 56.9 ± 28.6 ng/ml) compared to those with CC genotype (mean ± SD, 29.5 ± 21.1 ng/ml; p = 0.001) and those with CT genotype (mean ± SD, 25.7 ± 18.8; p = 0.002). Besides the level of serum TGF-β1 in patients was found to be lower than in control subjects (Figure 1), significantly higher TGF-β1 levels were observed in SLE patients having the TT genotype (mean ± SD, 9.8 ± 2.6 ng/ml) compared to patients with CC genotype (mean ± SD, 6.7 ± 2.6 ng/ ml; p=0.023). Overall, individuals with TT homozygous genotype had higher serum TGF-β1 concentration in comparison to those with either CC homozygous genotype or CT heterozy‐ gous genotype.

**4. Role of IL-10 and TGF-β1 genotypes as risk factor for appearance of SLE**

Gene Polymorphisms of Immunoregulatory Cytokines IL-10 and TGF-β1 in Systemic Lupus Erythematosus

http://dx.doi.org/10.5772/53799

41

**4.1. Role of IL-10 promoter polymorphism in susceptibility and clinical manifestation of**

Several evidences suggest that IL-10 could be a strong candidate gene influencing SLE susceptibility. IL-10 gene has been mapped to chromosome 1q31-32, which is a susceptibility region for SLE (LOD=3.79) [Johanneson et al., 2002]. It is also homologous to a murine SLE susceptibility region [Tsao et al., 1997]. More recently, Gateva et al., 2010 performed a largescale replication study involving 1,310 cases and 7,859 controls, and identified 21 additional

However, in spite of the considerable number of genetic studies performed, no definitive result about its involvement in SLE susceptibility was achieved. Some works showed significant associations between IL-10 microsatellites or SNPs with SLE susceptibility or with the development of certain clinical or immunological features [Rood et al., 1999; D'Alfonso et al., 2002; Schotte et al., 2004; Chen et al., 2006; Sung et al., 2006; Chong et al., 2006; Rosado et al., 2008] while other studies indicated that these polymorphisms did not appear to have any relevance in the disease [Alarcón-Riquelme et al., 1999; Van der Linden et al., 2000; Dijstel‐ bloem et al., 2002; Guarnizo-Zuccardi et al., 2007]. The role of IL-10 genotypes has been recently reviewed by Lopez et al., 2010. The more recent association studies dealing IL-10 promoter

With respect to microsatellite variants, different alleles of IL10.G have been reported to be associated with SLE incidence in various populations. Thus, frequency of IL10.G9 allele (21 CA repeats) was significantly decreased in European [D'Alfonso et al., 2000, D'Alfonso et al., 2002, Eskdale et al., 1997] and Mexican-American [Mehrian et al., 1998] SLE patients, whereas the long alleles IL10.G10, G11 and G13 (with a CA repeat number greater than 21) were significantly increased in Mexican-American [Mehrian et al., 1998], Italian [D'Alfonso et al., 2000, D'Alfonso et al., 2002] and British [Eskdale et al., 1997] patients respectively. On the contrary, an increase in IL10.G4 (short allele) was reported in Chinese patients [Chong et al., 2004] whereas no significant differences in IL10.G alleles were detected in other cohorts [Schotte et al., 2004, Alarcon-Riquelme et al., 1999; Johansson et al., 2002]. Recently, a large meta-analysis summarized the results focused on the role of IL-10 promoter polymorphisms for SLE susceptibility from 16 published case-control studies involving a total of 2391 SLE patients and 3483 controls (Nath et al., 2005). The results of the meta-analysis performed by Nath et al., 2005 showed a significant association between SLE and the G11 allele of IL10.G (OR=1.279, 95% CI; 1.027±1.593, P=0.028) in whole populations, and IL-10 promoter -1082G allele was associated with SLE in Asians (OR=1.358, 95% CI; 1.015±1.816, P=0.039). It has been reported that LPS-stimulated cells from individuals carriers of the IL10.G allele with 26 CA repeats presented higher IL-10 production than those from carriers of short alleles [Eskdale et al., 1998], suggesting that long alleles might be responsible for a high IL-10 production. Thus, accordingly to these data, high IL-10 producer genotypes (with more than 21 CA repeats) could be associated with SLE susceptibility, while presence of short alleles could confer a protective

candidate susceptibility loci for SLE. Among the newly identified SLE loci is IL-10.

polymorphisms for SLE susceptibility are summarized on Table 2.

effect [Chen et al., 2006; D'Alfonso et al., 2002].

**SLE**

The study of Awad and colleagues related the carriage of GG homozygous genotype in position +915 of TGFB1 to significantly higher TGF-β1 production in peripheral blood leukocytes [Awad et al., 1998]. However, Lu et al., 2004 also, analyzing the serum levels of TGF-β1 as well as the TGF-β1 production of unstimulated and stimulated peripheral blood mononuclear cells, did not observe functional correlations with TGF-β1 production and -509 and codon10 alleles. The authors raise the question whether the lower serum TGF-β1 level that cause defective immune regulation in SLE is primarily under genetic control or secondary to the influence of ongoing cellular interactions in the cytokine context. The data from our preliminary study shown that a number of factors such as age, sex, presence of disease, and allele variants of -509 C/T SNP in the gene for TGF-β1 influence the level of this cytokine in the serum. Thus, a genetically controlled low production of TGF-β1 is a predisposing factor for the loss of negative regulation found in SLE and may constitute an important component of the genetically determined susceptibility to this disease and the autoimmune events responsible for its pathogenesis, developed under appropriate environmental stimuli.

**Figure 2.** Mean (± SD) serum *TGF*-β1 concentrations (ng/ml) in SLE patients and healthy controls according to the gen‐ otypes of -509 C/T polymorphism of *TGFB1*. \* p < 0.05, \*\* p <.01

#### **4. Role of IL-10 and TGF-β1 genotypes as risk factor for appearance of SLE**

ng/ml; p = 0.001) and those with CT genotype (mean ± SD, 25.7 ± 18.8; p = 0.002). Besides the level of serum TGF-β1 in patients was found to be lower than in control subjects (Figure 1), significantly higher TGF-β1 levels were observed in SLE patients having the TT genotype (mean ± SD, 9.8 ± 2.6 ng/ml) compared to patients with CC genotype (mean ± SD, 6.7 ± 2.6 ng/ ml; p=0.023). Overall, individuals with TT homozygous genotype had higher serum TGF-β1 concentration in comparison to those with either CC homozygous genotype or CT heterozy‐

The study of Awad and colleagues related the carriage of GG homozygous genotype in position +915 of TGFB1 to significantly higher TGF-β1 production in peripheral blood leukocytes [Awad et al., 1998]. However, Lu et al., 2004 also, analyzing the serum levels of TGF-β1 as well as the TGF-β1 production of unstimulated and stimulated peripheral blood mononuclear cells, did not observe functional correlations with TGF-β1 production and -509 and codon10 alleles. The authors raise the question whether the lower serum TGF-β1 level that cause defective immune regulation in SLE is primarily under genetic control or secondary to the influence of ongoing cellular interactions in the cytokine context. The data from our preliminary study shown that a number of factors such as age, sex, presence of disease, and allele variants of -509 C/T SNP in the gene for TGF-β1 influence the level of this cytokine in the serum. Thus, a genetically controlled low production of TGF-β1 is a predisposing factor for the loss of negative regulation found in SLE and may constitute an important component of the genetically determined susceptibility to this disease and the autoimmune events responsible for its pathogenesis, developed under appropriate environmental stimuli.

CC 29,5 6,7 CT 25,7 8,3 TT 56,9 9,8

\*\*

40 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

otypes of -509 C/T polymorphism of *TGFB1*. \* p < 0.05, \*\* p <.01

controls (n=52) SLE (n=48)

**Figure 2.** Mean (± SD) serum *TGF*-β1 concentrations (ng/ml) in SLE patients and healthy controls according to the gen‐

\*

gous genotype.

**TGF-beta1 ng/ml**

#### **4.1. Role of IL-10 promoter polymorphism in susceptibility and clinical manifestation of SLE**

Several evidences suggest that IL-10 could be a strong candidate gene influencing SLE susceptibility. IL-10 gene has been mapped to chromosome 1q31-32, which is a susceptibility region for SLE (LOD=3.79) [Johanneson et al., 2002]. It is also homologous to a murine SLE susceptibility region [Tsao et al., 1997]. More recently, Gateva et al., 2010 performed a largescale replication study involving 1,310 cases and 7,859 controls, and identified 21 additional candidate susceptibility loci for SLE. Among the newly identified SLE loci is IL-10.

However, in spite of the considerable number of genetic studies performed, no definitive result about its involvement in SLE susceptibility was achieved. Some works showed significant associations between IL-10 microsatellites or SNPs with SLE susceptibility or with the development of certain clinical or immunological features [Rood et al., 1999; D'Alfonso et al., 2002; Schotte et al., 2004; Chen et al., 2006; Sung et al., 2006; Chong et al., 2006; Rosado et al., 2008] while other studies indicated that these polymorphisms did not appear to have any relevance in the disease [Alarcón-Riquelme et al., 1999; Van der Linden et al., 2000; Dijstel‐ bloem et al., 2002; Guarnizo-Zuccardi et al., 2007]. The role of IL-10 genotypes has been recently reviewed by Lopez et al., 2010. The more recent association studies dealing IL-10 promoter polymorphisms for SLE susceptibility are summarized on Table 2.

With respect to microsatellite variants, different alleles of IL10.G have been reported to be associated with SLE incidence in various populations. Thus, frequency of IL10.G9 allele (21 CA repeats) was significantly decreased in European [D'Alfonso et al., 2000, D'Alfonso et al., 2002, Eskdale et al., 1997] and Mexican-American [Mehrian et al., 1998] SLE patients, whereas the long alleles IL10.G10, G11 and G13 (with a CA repeat number greater than 21) were significantly increased in Mexican-American [Mehrian et al., 1998], Italian [D'Alfonso et al., 2000, D'Alfonso et al., 2002] and British [Eskdale et al., 1997] patients respectively. On the contrary, an increase in IL10.G4 (short allele) was reported in Chinese patients [Chong et al., 2004] whereas no significant differences in IL10.G alleles were detected in other cohorts [Schotte et al., 2004, Alarcon-Riquelme et al., 1999; Johansson et al., 2002]. Recently, a large meta-analysis summarized the results focused on the role of IL-10 promoter polymorphisms for SLE susceptibility from 16 published case-control studies involving a total of 2391 SLE patients and 3483 controls (Nath et al., 2005). The results of the meta-analysis performed by Nath et al., 2005 showed a significant association between SLE and the G11 allele of IL10.G (OR=1.279, 95% CI; 1.027±1.593, P=0.028) in whole populations, and IL-10 promoter -1082G allele was associated with SLE in Asians (OR=1.358, 95% CI; 1.015±1.816, P=0.039). It has been reported that LPS-stimulated cells from individuals carriers of the IL10.G allele with 26 CA repeats presented higher IL-10 production than those from carriers of short alleles [Eskdale et al., 1998], suggesting that long alleles might be responsible for a high IL-10 production. Thus, accordingly to these data, high IL-10 producer genotypes (with more than 21 CA repeats) could be associated with SLE susceptibility, while presence of short alleles could confer a protective effect [Chen et al., 2006; D'Alfonso et al., 2002].

observed that only GCC/GCC genotype was significant more frequent in SLE. Hence, the conclusion suggests the GCC/GCC promoter genotype may contribute to SLE incidence in Polish patients. A very recent study investigated the association between of IL-10 promoter polymorphisms (-1082, -819 and -592) with SLE in a total of 172 Taiwanese patients and 215 controls reported an association of IL-10 ATA haplotype with SLE in Taiwanese population [Lin et al., 2010]. In another Asian population, an association of ACC/ACC haplotype of IL-10

Gene Polymorphisms of Immunoregulatory Cytokines IL-10 and TGF-β1 in Systemic Lupus Erythematosus

Inthis regard,weinvestigatedtheroleof-1082A/GpromoterpolymorphismofIL-10geneas risk factor for development and clinical manifestations of SLE in Bulgarian population. Our prelimi‐ nary results did not reveal a significant association of -1082 SNP in IL-10 with SLE [Miteva et al., 2010]. New data for the distribution and the frequencies of the -1082A/G alleles and genotypes among the SLE patients and healthy controls are presented on Table 3. The results of our casecontrol studybasedon157patientswithSLEand166unaffectedcontrolindividuals showedthat the genotype distribution is consistent with those published for other Caucasian type control cohorts [Lopez et al., 2010). We also found the prevalence of homozygous GG genotype in SLE cases (27%) compared to the controls (13%) with OR = 1.185 (95% CI = 0.58÷2.45), although the difference did not reach statistical significance (p=0.548). In addition, we observed an in‐ creased frequency of GG genotype compared to the reference AA genotype in patients with antiphospholipid synrom (APS) (27%) compared with patients without APS (14%) with OR = 2.750, 95% CI = 0.910 ÷ 8.347, p = 0.074). This suggests that carriage of a higher IL-10 producing genotype is a risk factor for antiphospholipid autoantibody production and APS appearance,

**Genotype Allele**

**GG n (%)**

χ2 =0.203

p=0.934

**A n (%)**

http://dx.doi.org/10.5772/53799

43

**G n (%)**

201 131

in susceptibility to SLE has been observed by Hirankarn et al., 2006.

thus having a modifying effect on the clinical presentation of the disease.

**AG+AA n (%)**

59 83 142 107 24

**AG+GG n (%)**

56 74 130 101 27 186 128 (36%) (47%) (73%) (74%) (27%) (59%) (41%)

(36%) (50%) (86%) (64%) (14%) (61%) (39%)

1*.* 0.939 - 0.994 1.185 1 1.056 *ref* (0.56±1.57) (0.61±1.61) (0.58±2.42) *ref.* (0.76±1.47)

*p 0.799 - 0.981 0.614 0.735*

**Table 3.** Genotypic and allelic frequency of gene polymorphism at position -1082 A/G in IL-10 gene in SLE patients

0.844 0.793 0.814 1 0.947 1 (0.41±1.72) (0.40±1.56) (0.43±1.54) - *ref.* (0.68±1.31) *ref. p 0.614 0.471 0.5 - 0.735*

**AG n (%)**

**rs1800896-10 82 A/G IL10**

**SLE (n=157)**

**HC (n=166)**

OR (95% CI)

OR (95% CI)

and controls

**AA n (%)**

**Table 2.** Association of IL-10 promotor polymorphisms - IL10G, IL10R, -1082G/A (rs1800896), -819C/T (rs1800871), -592A/C (rs1800872), with SLE. Association studies published after 2005 years are given only.

Conflicting results were also obtained after examining the possible association between SLE susceptibility and SNPs at -1082, -819, and -592 positions of IL-10 gene in the different populations in which they were investigated. The frequency of high IL-10 producers (carriers of -1082G allele or GCC haplotype) has been found to be increased in several works with Asian [Nath et al., 2005; Hirankarn et al., 2006] or European [Rosado o et al., 2008; Sobkowiak et al., 2009] patients, although most of the studies performed in Caucasian populations did not show significant associations [Lazarus et al. 1997, Guarnizo-Zuccardi et al, 2007;, Koss et al., 2000; Suárez et al., 2005; Dijstelbloem et al., 2002; Van der Linden et al., 2000; Crawley et al., 1999].

Rosado et al., 2008 found that the GCC haplotype frequency was significantly higher in Spanish patients with SLE. To assess the functional role of genotypes, they also quantified serum IL-10 levels from patients and controls and found higher serum IL-10 levels in patients. On the basis of these data, they suggest that the IL-10 promoter haplotype that produces higher levels of cytokine is associated with SLE in Spanish population. Similar are the results and final conclusion of the study performed by Sobkowiak et al., 2009; despite the higher prevalence of the GCC/GCC, GCC/ATA and ATA/ATA genotypes in SLE patients than in controls, they observed that only GCC/GCC genotype was significant more frequent in SLE. Hence, the conclusion suggests the GCC/GCC promoter genotype may contribute to SLE incidence in Polish patients. A very recent study investigated the association between of IL-10 promoter polymorphisms (-1082, -819 and -592) with SLE in a total of 172 Taiwanese patients and 215 controls reported an association of IL-10 ATA haplotype with SLE in Taiwanese population [Lin et al., 2010]. In another Asian population, an association of ACC/ACC haplotype of IL-10 in susceptibility to SLE has been observed by Hirankarn et al., 2006.

**Polymoprhisms Association SLE/contr. Population References**

2391/3483 Meta-analysis Nath et al., 2005

350/330 Korean Sung et al., 2005

237/304 Taiwanese Chen et al., 2006

195/159 Thai Hirankarn et al.,

116/51 Spanish Rosado et al., 2008

No association 110/138 Chinese Yu et al., 2010

2006

Guarnizo-Zuccardi et al., 2007

2009

Association of IL10.G11 allele in whole populations; Association of -1082G allele with SLE in Asians

42 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

No association with susceptibility Association of -592C with SLE activity

Increased G9 and decreased G8 in SLE Association of G13 with ACLA-IgM Association of G8 with CNS lupus

Association of ACC/ACC with susceptibility to SLE

No association of microsatellites Increased GCC in SLE

**Table 2.** Association of IL-10 promotor polymorphisms - IL10G, IL10R, -1082G/A (rs1800896), -819C/T (rs1800871),

Conflicting results were also obtained after examining the possible association between SLE susceptibility and SNPs at -1082, -819, and -592 positions of IL-10 gene in the different populations in which they were investigated. The frequency of high IL-10 producers (carriers of -1082G allele or GCC haplotype) has been found to be increased in several works with Asian [Nath et al., 2005; Hirankarn et al., 2006] or European [Rosado o et al., 2008; Sobkowiak et al., 2009] patients, although most of the studies performed in Caucasian populations did not show significant associations [Lazarus et al. 1997, Guarnizo-Zuccardi et al, 2007;, Koss et al., 2000; Suárez et al., 2005; Dijstelbloem et al., 2002; Van der Linden et al., 2000; Crawley et al., 1999].

Rosado et al., 2008 found that the GCC haplotype frequency was significantly higher in Spanish patients with SLE. To assess the functional role of genotypes, they also quantified serum IL-10 levels from patients and controls and found higher serum IL-10 levels in patients. On the basis of these data, they suggest that the IL-10 promoter haplotype that produces higher levels of cytokine is associated with SLE in Spanish population. Similar are the results and final conclusion of the study performed by Sobkowiak et al., 2009; despite the higher prevalence of the GCC/GCC, GCC/ATA and ATA/ATA genotypes in SLE patients than in controls, they

IL10G, IL10R -1082; -819; -592

IL10G

IL10G, IL10R -1082; -819; -592

Inthis regard,weinvestigatedtheroleof-1082A/GpromoterpolymorphismofIL-10geneas risk factor for development and clinical manifestations of SLE in Bulgarian population. Our prelimi‐ nary results did not reveal a significant association of -1082 SNP in IL-10 with SLE [Miteva et al., 2010]. New data for the distribution and the frequencies of the -1082A/G alleles and genotypes among the SLE patients and healthy controls are presented on Table 3. The results of our casecontrol studybasedon157patientswithSLEand166unaffectedcontrolindividuals showedthat the genotype distribution is consistent with those published for other Caucasian type control cohorts [Lopez et al., 2010). We also found the prevalence of homozygous GG genotype in SLE cases (27%) compared to the controls (13%) with OR = 1.185 (95% CI = 0.58÷2.45), although the difference did not reach statistical significance (p=0.548). In addition, we observed an in‐ creased frequency of GG genotype compared to the reference AA genotype in patients with antiphospholipid synrom (APS) (27%) compared with patients without APS (14%) with OR = 2.750, 95% CI = 0.910 ÷ 8.347, p = 0.074). This suggests that carriage of a higher IL-10 producing genotype is a risk factor for antiphospholipid autoantibody production and APS appearance, thus having a modifying effect on the clinical presentation of the disease.

**Table 3.** Genotypic and allelic frequency of gene polymorphism at position -1082 A/G in IL-10 gene in SLE patients and controls

The presence of autoantibodies, mainly directed against nuclear antigens (ANAs), is one of the most characteristic features of SLE. The effect of IL-10 genotypes did not seem to be especially relevant, although it has been reported an increased prevalence of antibod‐ ies against several extractable nuclear antigens (anti-ENA) in patients with the allele IL-10.G9 [Eskdale, et al., 1997], and the presence of anti-Sm antibodies was found signifi‐ cantly overrepresented among patient carriers of G14 and G15 alleles and R2-G15 and R2- G14 haplotypes [Schotte et al., 2004]. An association of the carriage of low IL-10 producer alleles such as IL10.G13 allele with the presence of anticardiolipin IgM antibodies and IL10.G8 allele with neurological affectation has been reported in Taiwanese patients with SLE [Chen et al., 2006], results very similar to the data from our study.

**4.2. Role of TGB-β1 genetic polymorphisms in susceptibility and clinical manifestation of**

Gene Polymorphisms of Immunoregulatory Cytokines IL-10 and TGF-β1 in Systemic Lupus Erythematosus

Regarding the association between decreased *TGF*-β1 serum levels and the development of autoimmunity, the mechanisms which control the concentration of *TGF*-β1 in plasma are under extensive investigations. The concentration of both latent complex and active *TGF*-β1 in plasma has been shown to be predominantly under genetic control [Grainger et al., 1999]. In light of these findings TGF-β1 gene is a functional candidate gene for genetic predisposition in

The presence of polymorphisms in the *TGFB1* locus may indicate predisposition to diseases, such as systemic lupus erythematosus that have been linked here and elsewhere [Caserta et al., 2004; Lu et al., 2004] to the circulating levels of TGF-β1. To test this hypothesis, we performed a population based case-control study to investigate the association of -509C/T polymorphism of the *TGFB1* gene with susceptibility to SLE [Manolova et al., 2012]. In this investigation, the change at position -509C/T in the *TGFB1* gene (rs1800469) was studied using RFLP-PCR among 147 cases with SLE and 134 normal Bulgarian subjects. The genotype distribution and allele frequencies of -509C/T SNP in gene promoter of *TGFB1* among SLE

> **CT+TT n (%)**

48 79 127 101 22 175 123

48 57 105 86 29 153 115

(35.8%) (42.5%) (78.3%) (64.1%) (21.6%) (57.1%) (42.9%)

(0.63-2.77) (0.91±3.69) (0.83±3.07) *ref.* (0.76±1.5) *ref.*

1 1.386 - 1.17 0.759 1 0.935

*ref.* (0.79±2.43) (0.7±1.98) (0.36±1.59) *ref.* (0.66±1.33)

**Genotype Allele**

**TT n (%)**

> χ2=3.735 p=0.155

1 1.069 1

**C n (%)**

http://dx.doi.org/10.5772/53799

45

**T n (%)**

(58.7%) (41.3%)

**SLE**

**rs1800469 -509C/T TGFB1**

SLE (n=149)

HC (n=134)

OR (95% CI)

OR (95% CI)

controls

systemic lupus erythematosus.

**CC n (%)**

patients and healthy donors are presented in Table 4.

**CT n (%)**

*1.318* 1.827 1.59

**CT+CC n (%)**

(32.2%) (53%) (85.2%) (67.8%) (14.8%))

*p 0.428 0.068 0.133 - 0.694*

*p 0.223 - 0.522 0.428 0.694*

**Table 4.** Genotypic and allelic frequency of gene polymorphism at position -509 in *TGFB1* gene in SLE patients and

Considering increased circulating levels of IL-10 have been consistently reported in the sera of patients with SLE, it is possible that different cytokine production may not only influence the autoantibody production, but also the clinical presentation of the disease. However, there were no definitive data on the association of IL-10 polymorphisms and specific clinical manifestations, probably due to the heterogeneity of the disease. For instance, renal involvement has been associated with both high (GCC) [Lazarus et al., 1997, Zhu et al., 2005] and low (ATA) [Mok et al., 1999] IL-10 producer genotypes. High prevalence of neuropsychiatric [Rood et al., 1999; Chen et al. 2006] and cardiovascular disorders [Fei et al., 2004] has been reported in patients with low genetic production whereas high IL-10 production has been linked to an increased incidence of serositis, hematological disorder [Chong et al.], SLICC/ACR Damage Index [Sung et al., 2006] and presence of discoid or mucocutaneous lesions [Suárez et al., 2005; Alarcón-Riquelme et al.,1999]. This last association was supported by the increased frequency of the high producer -1082G allele observed in patients with discoid lupus erythematosus [Suárez et al., 2005; Van der Linden et al., 2000] and by the fact that cutaneous manifestations improved in SLE patients under anti IL-10 monoclonal antibody treatment [Llorente et al., 2000]. In conclusion IL-10 promoter SNPs alone have not exhibits strong association with SLE susceptibility, but their role cant' be excluded. Each polymorphism in regulatory regions of gene, may either directly influence gene expression or indirectly via tight linkage with other polymor‐ phisms occurring elsewhere in the same or in other cytokine gene. A particular combina‐ tion of SNPs on cytokine genes in individual genotype has different impacts on induced cytokine production. Miteva and Stanilova investigate the combined effect of -1082A\*G in IL10 and +16974A\*C in IL12B SNPs on induced cytokine production by stimulated peripheral blood mononuclear cells isolated from healthy donors (Miteva, Stanilova, 2008). Results demonstrated that the production of IL-10 from PBMC depended on both, -1082А\*G in IL10 and +16974A\*C in IL12B polymorphisms and the presence of high producer IL-12p40 genotype led to diminished production of IL-10 determined by -1082\*Gallele of SNP in IL10. In the same vein, we suppose that individuals with genotype which combine SNPs responsible for higher production of IL-10 simultaneously with lower production of TGF-β1 should be more susceptible to SLE.

#### **4.2. Role of TGB-β1 genetic polymorphisms in susceptibility and clinical manifestation of SLE**

The presence of autoantibodies, mainly directed against nuclear antigens (ANAs), is one of the most characteristic features of SLE. The effect of IL-10 genotypes did not seem to be especially relevant, although it has been reported an increased prevalence of antibod‐ ies against several extractable nuclear antigens (anti-ENA) in patients with the allele IL-10.G9 [Eskdale, et al., 1997], and the presence of anti-Sm antibodies was found signifi‐ cantly overrepresented among patient carriers of G14 and G15 alleles and R2-G15 and R2- G14 haplotypes [Schotte et al., 2004]. An association of the carriage of low IL-10 producer alleles such as IL10.G13 allele with the presence of anticardiolipin IgM antibodies and IL10.G8 allele with neurological affectation has been reported in Taiwanese patients with

Considering increased circulating levels of IL-10 have been consistently reported in the sera of patients with SLE, it is possible that different cytokine production may not only influence the autoantibody production, but also the clinical presentation of the disease. However, there were no definitive data on the association of IL-10 polymorphisms and specific clinical manifestations, probably due to the heterogeneity of the disease. For instance, renal involvement has been associated with both high (GCC) [Lazarus et al., 1997, Zhu et al., 2005] and low (ATA) [Mok et al., 1999] IL-10 producer genotypes. High prevalence of neuropsychiatric [Rood et al., 1999; Chen et al. 2006] and cardiovascular disorders [Fei et al., 2004] has been reported in patients with low genetic production whereas high IL-10 production has been linked to an increased incidence of serositis, hematological disorder [Chong et al.], SLICC/ACR Damage Index [Sung et al., 2006] and presence of discoid or mucocutaneous lesions [Suárez et al., 2005; Alarcón-Riquelme et al.,1999]. This last association was supported by the increased frequency of the high producer -1082G allele observed in patients with discoid lupus erythematosus [Suárez et al., 2005; Van der Linden et al., 2000] and by the fact that cutaneous manifestations improved in SLE patients under anti IL-10 monoclonal antibody treatment [Llorente et al., 2000]. In conclusion IL-10 promoter SNPs alone have not exhibits strong association with SLE susceptibility, but their role cant' be excluded. Each polymorphism in regulatory regions of gene, may either directly influence gene expression or indirectly via tight linkage with other polymor‐ phisms occurring elsewhere in the same or in other cytokine gene. A particular combina‐ tion of SNPs on cytokine genes in individual genotype has different impacts on induced cytokine production. Miteva and Stanilova investigate the combined effect of -1082A\*G in IL10 and +16974A\*C in IL12B SNPs on induced cytokine production by stimulated peripheral blood mononuclear cells isolated from healthy donors (Miteva, Stanilova, 2008). Results demonstrated that the production of IL-10 from PBMC depended on both, -1082А\*G in IL10 and +16974A\*C in IL12B polymorphisms and the presence of high producer IL-12p40 genotype led to diminished production of IL-10 determined by -1082\*Gallele of SNP in IL10. In the same vein, we suppose that individuals with genotype which combine SNPs responsible for higher production of IL-10 simultaneously with lower

SLE [Chen et al., 2006], results very similar to the data from our study.

44 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

production of TGF-β1 should be more susceptible to SLE.

Regarding the association between decreased *TGF*-β1 serum levels and the development of autoimmunity, the mechanisms which control the concentration of *TGF*-β1 in plasma are under extensive investigations. The concentration of both latent complex and active *TGF*-β1 in plasma has been shown to be predominantly under genetic control [Grainger et al., 1999]. In light of these findings TGF-β1 gene is a functional candidate gene for genetic predisposition in systemic lupus erythematosus.

The presence of polymorphisms in the *TGFB1* locus may indicate predisposition to diseases, such as systemic lupus erythematosus that have been linked here and elsewhere [Caserta et al., 2004; Lu et al., 2004] to the circulating levels of TGF-β1. To test this hypothesis, we performed a population based case-control study to investigate the association of -509C/T polymorphism of the *TGFB1* gene with susceptibility to SLE [Manolova et al., 2012]. In this investigation, the change at position -509C/T in the *TGFB1* gene (rs1800469) was studied using RFLP-PCR among 147 cases with SLE and 134 normal Bulgarian subjects. The genotype distribution and allele frequencies of -509C/T SNP in gene promoter of *TGFB1* among SLE patients and healthy donors are presented in Table 4.

**Table 4.** Genotypic and allelic frequency of gene polymorphism at position -509 in *TGFB1* gene in SLE patients and controls

The genotype distribution for TGFB1 -509C/T polymorphism was in agreement with Hardy-Weinberg equilibrium among cases (χ2=2.00; p=0.367) and controls (χ2=2.237; p=0.326). Homozygous CC genotype was found in 32.2% of patients and 36.8% of control subjects, heterozygous CT genotype was observed in 53% of SLE patients and 42.5% of controls, homozygous TT genotype was detected in 14.8% of cases and 21.6% of controls. There were no significant differences in the genotype (p=0.155) and allele (p=0.694) frequencies of -509C/T polymorphism of the TGFB1 gene between SLE patients and controls. However, we observed a higher frequency of heterozygous CT genotype (OR = 1.827; 95% CI 0.91±3.69; p = 0.068) and lower frequency of TT genotype (OR = 0,759; 95% CI: 0.36±1.59; p=0.428) in SLE patients compared to healthy controls. In logistic regression analysis the presence of allele C in the genotype (CT + CC versus TT) was associated with a 1.6 times higher risk of developing systemic lupus erythematosus.

**Polymoprhisms Association SLE/contr. Population References** +915G/C (rs1800471) No association 203/158 German Schotte et al., 2003

Gene Polymorphisms of Immunoregulatory Cytokines IL-10 and TGF-β1 in Systemic Lupus Erythematosus

Association of +869C allele with SLE; decreased high TGF-β1 producers haplotypes in SLE (codon10 T allele/25 G allele) No association with clinical manifestations

+869TT associated with aseptic necrosis and anti-Ro antibodies

(OR = 0,759; 95% CI: 0.36±1.59) allele C (CT+CC vs TT) risk factor for SLE (OR=1.59; 95% CI: 0.83±3.07)

Lu et al. conducted a case-control study involving 134 patients and 182 healthy individuals of Taiwanese origin to evaluate the association of -509C/T SNP in *TGFB1* with susceptibility to systemic lupus erythematosus [Lu et al., 2004]. In the same study, authors investigated also association of some others TGFβ1 single nucleotide polymorphisms, including -988C/A, -800G/A, +869T/C (Leu10Pro), and +915 G/C (Arg25Pro) with susceptibility to SLE and shown that none of the TGFβ1 SNPs was strongly associated with SLE in Taiwanese patients. They conclude that these polymorphisms do not represent a genetic predisposition to SLE. In another study, Schotte and colleagues investigated the +915G/C polymorphism at codon25 and did not found any association between this polymorphism and SLE in German population [Schotte et al., 2003]. In addition, authors found no association of major disease manifestations or specific autoantibodies with TGFB1 genotypes or alleles. The authors conclude that +915G/ C polymorphism in TGFB1 neither significantly contributes to the disease susceptibility, nor predisposes to clinical and immunological manifestations typical of SLE. Also, there were no significant associations between several SNPs from the TGFβ1 including -509C/T, +869T/C,

+869T/C (rs1982073) No association with susceptibility

**Table 6.** Association of TGF-β1 gene polymorphisms with SLE.

No association 138/182 Taiwanese Lu et al., 2004

No association 272/307 Sweden Vuong et al., 2010

America

120/102 Colombian Guarnizo-Zuccardi et

196/102 Japan Wang et al., 2007

149/134 Bulgarian Manolova et al. 2012

Caserta et al., 2004

http://dx.doi.org/10.5772/53799

47

al., 2007

+869T/C (rs1982073), +915G/C (rs1800471)

Genotype and allele frequencies of -509C/T polymorphism in *TGFB1* which we establish‐ ed in Bulgarian population were comparable to those found in other populations. Howev‐ er, the available data in the literature reveal the existence of ethnic differences in frequencies of the allele variants of this polymorphic marker (Table 5). C allele had the higher representation among Europeans in French [Cambien et al., 1996], German [Wu et al., 2008] and British [Awad et al., 1998] studies with a frequency of 65 to 76 percent, while among healthy individuals from different Asian ethnicities T allele occurs more frequent‐ ly or almost equally with the C allele [Amirghofran Z, 2009; Zhang et al., 2009; Chung et al.,, 2007]. In Bulgarian population allele C is slightly more common and was found in 57% of healthy subjects and in 59% of cases with SLE.

**Table 5.** Interethnic differences in allele frequencies of -509C/T SNP in *TGFB1* in healthy controls.

According to our knowledge there are only a limited number of studies aiming to evaluate the possible role of polymorphisms in the TGF-β1 gene as predisposing factors for SLE, but the results of these studies are contradictory. The polymorphisms in of -509C/T SNP in TGF-β1 have been explored in SLE only by three research teams [Lu et al., 2004; Caserta et al., 2004; Vuong et al., 2010]. In overall, the contradictory results in the literature could be explained by the genetic heterogeneity of SLE in different populations and possible sample stratification. Table 6 summarizes the association studies dealing TGF-β1 gene polymorphisms for SLE susceptibility.

Gene Polymorphisms of Immunoregulatory Cytokines IL-10 and TGF-β1 in Systemic Lupus Erythematosus http://dx.doi.org/10.5772/53799 47

**Table 6.** Association of TGF-β1 gene polymorphisms with SLE.

The genotype distribution for TGFB1 -509C/T polymorphism was in agreement with Hardy-Weinberg equilibrium among cases (χ2=2.00; p=0.367) and controls (χ2=2.237; p=0.326). Homozygous CC genotype was found in 32.2% of patients and 36.8% of control subjects, heterozygous CT genotype was observed in 53% of SLE patients and 42.5% of controls, homozygous TT genotype was detected in 14.8% of cases and 21.6% of controls. There were no significant differences in the genotype (p=0.155) and allele (p=0.694) frequencies of -509C/T polymorphism of the TGFB1 gene between SLE patients and controls. However, we observed a higher frequency of heterozygous CT genotype (OR = 1.827; 95% CI 0.91±3.69; p = 0.068) and lower frequency of TT genotype (OR = 0,759; 95% CI: 0.36±1.59; p=0.428) in SLE patients compared to healthy controls. In logistic regression analysis the presence of allele C in the genotype (CT + CC versus TT) was associated with a 1.6 times higher risk of developing

Genotype and allele frequencies of -509C/T polymorphism in *TGFB1* which we establish‐ ed in Bulgarian population were comparable to those found in other populations. Howev‐ er, the available data in the literature reveal the existence of ethnic differences in frequencies of the allele variants of this polymorphic marker (Table 5). C allele had the higher representation among Europeans in French [Cambien et al., 1996], German [Wu et al., 2008] and British [Awad et al., 1998] studies with a frequency of 65 to 76 percent, while among healthy individuals from different Asian ethnicities T allele occurs more frequent‐ ly or almost equally with the C allele [Amirghofran Z, 2009; Zhang et al., 2009; Chung et al.,, 2007]. In Bulgarian population allele C is slightly more common and was found in 57%

**Frequency (%) among healthy controls**

**British study Awad et al., 1998**

**German study Wu et al., 2009**

**Asian study Lu et al., 2004**

systemic lupus erythematosus.

**Locus Allele**

susceptibility.

of healthy subjects and in 59% of cases with SLE.

46 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

**Bulgarian study Data from this study**

**French study Cambien et al., 1996**

**Table 5.** Interethnic differences in allele frequencies of -509C/T SNP in *TGFB1* in healthy controls.

According to our knowledge there are only a limited number of studies aiming to evaluate the possible role of polymorphisms in the TGF-β1 gene as predisposing factors for SLE, but the results of these studies are contradictory. The polymorphisms in of -509C/T SNP in TGF-β1 have been explored in SLE only by three research teams [Lu et al., 2004; Caserta et al., 2004; Vuong et al., 2010]. In overall, the contradictory results in the literature could be explained by the genetic heterogeneity of SLE in different populations and possible sample stratification. Table 6 summarizes the association studies dealing TGF-β1 gene polymorphisms for SLE

Т 43 34 24 30 52

Lu et al. conducted a case-control study involving 134 patients and 182 healthy individuals of Taiwanese origin to evaluate the association of -509C/T SNP in *TGFB1* with susceptibility to systemic lupus erythematosus [Lu et al., 2004]. In the same study, authors investigated also association of some others TGFβ1 single nucleotide polymorphisms, including -988C/A, -800G/A, +869T/C (Leu10Pro), and +915 G/C (Arg25Pro) with susceptibility to SLE and shown that none of the TGFβ1 SNPs was strongly associated with SLE in Taiwanese patients. They conclude that these polymorphisms do not represent a genetic predisposition to SLE. In another study, Schotte and colleagues investigated the +915G/C polymorphism at codon25 and did not found any association between this polymorphism and SLE in German population [Schotte et al., 2003]. In addition, authors found no association of major disease manifestations or specific autoantibodies with TGFB1 genotypes or alleles. The authors conclude that +915G/ C polymorphism in TGFB1 neither significantly contributes to the disease susceptibility, nor predisposes to clinical and immunological manifestations typical of SLE. Also, there were no significant associations between several SNPs from the TGFβ1 including -509C/T, +869T/C, intronic G/T (rs2241715), and3'-UTR A/G(rs6957) with SLE or with lupus nephritis in Sweden population [Vuong et al., 2010].

implicated in SLE development have been identified and was summarized in this review. SLE is clinically heterogeneous and genetically complex, and we expect that individual genes and cytokine patterns will be more or less important to different disease manifestations and subgroups of patients. Defining these genotype-cytokine-phenotype relationships will

Gene Polymorphisms of Immunoregulatory Cytokines IL-10 and TGF-β1 in Systemic Lupus Erythematosus

http://dx.doi.org/10.5772/53799

49

In ours and others studies have been analyzed the association of IL-10 and TGF-β1genetic variants with susceptibility to and outcome of SLE, showing variable results in most cases. However, it is known that the actions of cytokines may be profoundly conditioned by the presence of other cytokines, this being particularly true in the case of IL-10 and TGF-β1. New studies of particular combinations of SNPs on these cytokine genes in individual genotype and their impacts on the induced cytokine production could reveal the relation with susceptibility

and Spaska Stanilova3

3 Department of Molecular Biology, Immunology and Medical Genetics, Medical Faculty,

[1] Alarcón-Riquelme ME, Lindqvist AK, Jonasson I, Johanneson B, Sandino S, Alcocer-Varela J, Granados J, Kristjánsdóttir H, Gröndal G, Svenungsson E, Lundberg I, Steinsson K, Klareskog L, Sturfelt G, Truedsson L, Alarcón-Segovia D, Gyllensten UB. Genetic analysis of the contribution of IL10 to systemic lupus erythematosus. J

[2] Amirghofran, Z, Jalali, S. A, Ghaderi, A, & Hosseini, S. V. Genetic polymorphism in the transforming growth factor beta1 gene (-509 C/T and-800 G/A) and colorectal

[3] Aoki, C. A, Borchers, A. T, Li, M, Flavell, R. A, Bowlus, C. L, Ansari, A. A, & Gersh‐ win, M. E. Transforming growth factor beta (TGF-beta) and autoimmunity. Autoim‐

[4] Awad, M. R, Gamel, A, Hasleton, P, Turner, D. M, Sinnott, P. J, & Hutchinson, I. V. Genotypic variation in the transforming growth factor-beta1 gene: association with

1 Department of Health Care, Medical Faculty, Trakia University, Stara Zagora, Bulgaria

2 Clinic of Rheumatology, University Hospital, Medical University, Sofia, Bulgaria

increase our understanding of both initial and progression disease pathogenesis.

and clinical presentation of autoimmunity diseases.

, Mariana Ivanova2

Trakia University, Stara Zagora, Bulgaria

Rheumatol 1999;26(10):2148-52.

mun Rev (2005). , 4(7), 450-9.

cancer. Cancer Genet Cytogenet (2009). , 190(1), 21-5.

**Author details**

Irena Manolova1

**References**

In contrast to these data are the results obtained by Guarnizo-Zuccardi et al., 2007 for several cytokine gene polymorphisms in Colombian patients with SLE. They analyze the relation between the +868T/C and the +915G/C SNP in *TGFB1* with the development and clinical manifestations of SLE. The authors found a strong association of SLE with the TGFβ1 codon25 C allele, associated with decreased TGF-β1 production and found lower rates of higher-producing GG genotype and a higher frequency of heterozygous genotypes in this polymorphic marker in patients with SLE. As for the +868T/C polymorphism at codon10, Guarnizo-Zuccardi et al., 2007 did not observe association between SLE and codon10 when analyzing independently, but they found a significant association when the haplotypes codon10/20 were evaluated, which could be because of the linkage disequilibri‐ um between the two SNPs. This extended genotypic analysis revealed a lower frequency of high TGF-β1 producers – haplotype 10/25 T/T-G/G in Colombian patients with SLE. Unlike the relationship of +868T/C and +915G/C SNPs of the *TGFB1* gene to disease susceptibility, they found no association between clinical features of the disease and the polymorphisms studied. As opposed to this report, Wang et al., 2007 did not find an impact of +869T/C polymorphism in TGF-β1 gene on disease susceptibility in population-based case-control study involving 196 patients with SLE and 106 healthy controls in Japan. However, they found an association between +869T/C *TGFB1* polymorphism and several clinical features of SLE. The carriage of TT genotype of +869T/C polymorphism which is associated with a lower serum TGF-β1 level was related to the occurrence of aseptic necrosis and higher incidence of anti-SSA/Ro antibodies in SLE patients. Consistent with the last finding, the children with the TT +869T/C genotype of *TGFB1* gene have been reported to be more susceptible to anti-SSA/Ro antibody-associated congenital hear block [Clancy et al., 2003].

Systemic lupus erythematosus is a heterogeneous disease with diverse clinical manifestations that range could be due to genetic factors. In this regard, we also analyzed the effect of the -509C/T polymorphism in TGFB1 the clinical manifestations evolved in the course of the disease [Manolova et al., 2012]. The results of our study demonstrated a weak association of -509C/T polymorphism of TGFB1 with clinical manifestations of SLE. The carriage of the heterozygous genotype was associated with about 2-fold higher risk for the occurrence of hematological manifestations (OR=2.41; 95%CI: 1.10±5.32; p=0.016) and antibodies against dsDNA (OR = 2.0; 95% CI: 0.96±4.2, p = 0.045) in lupus patients, while the CC genotype is a protective factor for these events. Based on our and others data, we could assume the TGFβ1 gene polymorphisms as one of the genetic factors that explain the heterogeneity seen in SLE.

#### **5. Conclusions**

In recent years, efforts have been made to identify genes involved in the genetic predisposition and severity of SLE. During the last two decades, many of the 'candidate' cytokine genes implicated in SLE development have been identified and was summarized in this review. SLE is clinically heterogeneous and genetically complex, and we expect that individual genes and cytokine patterns will be more or less important to different disease manifestations and subgroups of patients. Defining these genotype-cytokine-phenotype relationships will increase our understanding of both initial and progression disease pathogenesis.

In ours and others studies have been analyzed the association of IL-10 and TGF-β1genetic variants with susceptibility to and outcome of SLE, showing variable results in most cases. However, it is known that the actions of cytokines may be profoundly conditioned by the presence of other cytokines, this being particularly true in the case of IL-10 and TGF-β1. New studies of particular combinations of SNPs on these cytokine genes in individual genotype and their impacts on the induced cytokine production could reveal the relation with susceptibility and clinical presentation of autoimmunity diseases.

#### **Author details**

intronic G/T (rs2241715), and3'-UTR A/G(rs6957) with SLE or with lupus nephritis in Sweden

In contrast to these data are the results obtained by Guarnizo-Zuccardi et al., 2007 for several cytokine gene polymorphisms in Colombian patients with SLE. They analyze the relation between the +868T/C and the +915G/C SNP in *TGFB1* with the development and clinical manifestations of SLE. The authors found a strong association of SLE with the TGFβ1 codon25 C allele, associated with decreased TGF-β1 production and found lower rates of higher-producing GG genotype and a higher frequency of heterozygous genotypes in this polymorphic marker in patients with SLE. As for the +868T/C polymorphism at codon10, Guarnizo-Zuccardi et al., 2007 did not observe association between SLE and codon10 when analyzing independently, but they found a significant association when the haplotypes codon10/20 were evaluated, which could be because of the linkage disequilibri‐ um between the two SNPs. This extended genotypic analysis revealed a lower frequency of high TGF-β1 producers – haplotype 10/25 T/T-G/G in Colombian patients with SLE. Unlike the relationship of +868T/C and +915G/C SNPs of the *TGFB1* gene to disease susceptibility, they found no association between clinical features of the disease and the polymorphisms studied. As opposed to this report, Wang et al., 2007 did not find an impact of +869T/C polymorphism in TGF-β1 gene on disease susceptibility in population-based case-control study involving 196 patients with SLE and 106 healthy controls in Japan. However, they found an association between +869T/C *TGFB1* polymorphism and several clinical features of SLE. The carriage of TT genotype of +869T/C polymorphism which is associated with a lower serum TGF-β1 level was related to the occurrence of aseptic necrosis and higher incidence of anti-SSA/Ro antibodies in SLE patients. Consistent with the last finding, the children with the TT +869T/C genotype of *TGFB1* gene have been reported to be more susceptible to anti-SSA/Ro antibody-associated congenital hear block

Systemic lupus erythematosus is a heterogeneous disease with diverse clinical manifestations that range could be due to genetic factors. In this regard, we also analyzed the effect of the -509C/T polymorphism in TGFB1 the clinical manifestations evolved in the course of the disease [Manolova et al., 2012]. The results of our study demonstrated a weak association of -509C/T polymorphism of TGFB1 with clinical manifestations of SLE. The carriage of the heterozygous genotype was associated with about 2-fold higher risk for the occurrence of hematological manifestations (OR=2.41; 95%CI: 1.10±5.32; p=0.016) and antibodies against dsDNA (OR = 2.0; 95% CI: 0.96±4.2, p = 0.045) in lupus patients, while the CC genotype is a protective factor for these events. Based on our and others data, we could assume the TGFβ1 gene polymorphisms as one of the genetic factors that explain the heterogeneity seen in SLE.

In recent years, efforts have been made to identify genes involved in the genetic predisposition and severity of SLE. During the last two decades, many of the 'candidate' cytokine genes

population [Vuong et al., 2010].

48 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

[Clancy et al., 2003].

**5. Conclusions**

Irena Manolova1 , Mariana Ivanova2 and Spaska Stanilova3

1 Department of Health Care, Medical Faculty, Trakia University, Stara Zagora, Bulgaria

2 Clinic of Rheumatology, University Hospital, Medical University, Sofia, Bulgaria

3 Department of Molecular Biology, Immunology and Medical Genetics, Medical Faculty, Trakia University, Stara Zagora, Bulgaria

#### **References**

transforming growth factor-beta1 production, fibrotic lung disease, and graft fibrosis after lung transplantation. Transplantation (1998). , 66(8), 1014-20.

tion: evidence for a significant association with interleukin-10. Arthritis Rheum

Gene Polymorphisms of Immunoregulatory Cytokines IL-10 and TGF-β1 in Systemic Lupus Erythematosus

http://dx.doi.org/10.5772/53799

51

[16] Alfonso, D, Rampi, S, Rolando, M, Giordano, V, & Momigliano-richiardi, M. New polymorphisms in the IL-10 promoter region. Genes Immun 2000;1:231-233., 2000.

[17] Dang, H, Geiser, A. G, Letterio, J. J, Nakabayashi, T, Kong, L, Fernandes, G, & Talal, N. SLE-like autoantibodies and Sjögren's syndrome-like lymphoproliferation in TGF-

[18] De Waal Malefyt RDYssel H, de Vries JE. Direct effects of IL-10 on subsets of human CD4+ T cell clones and resting T cells. Specific inhibition of IL-2 production and pro‐

[19] Dijstelbloem, H. M, Hepkema, B. G, et al. The R-H polymorphism of Fcγ receptor IIa as a risk factor for systemic lupus erythematosus is independent of single-nucleotide polymorphisms in the interleukin-10 gene promoter. Arthritis and Rheumatism

[20] Ding, L, Linsley, P. S, Huang, L-Y, Germain, R. N, & Shevach, E. M. IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7

[21] Diveu, C, Mcgeachy, M. J, & Cua, D. J. Cytokines that regulate autoimmunity. Curr

[22] Dobreva, Z. G, Miteva, L. D, & Stanilova, S. A. The inhibition of JNK and MAPKs downregulates IL-10 and differentially affects c-Jun gene expression in human mono‐

[23] Dubois, C. M, Laprise, M-H, Blanchette, F, Gentry, L. E, & Leduc, R. Processing of transforming growth factor β1 precursor by human furin convertase. J Biol Chem

[24] Eskdale J & Galager GA polymorphic dinucleotide repeat in the human IL- 10 pro‐

[25] Eskdale, J, Gallagher, G, Verweij, C. L, & Keijsers, V. Westendorp RGJ, and. Huizinga TWJ. Interleukin 10 secretion in relation to human IL-10 locus haplotypes. Proc Natl

[26] Eskdale, J, Kube, D, Tesch, H, & Gallagher, G. Mapping of the human IL10 gene and further characterization of the 5' flanking sequence. Immunogenetics (1997). , 46,

[27] Eskdale, J, Wordsworth, P, Bowman, S, Field, M, & Gallagher, G. Associations be‐ tween polymorphism at the human IL-10 locus and systemic lupus erythematosus.

expression. Journal of Immunology (1993). , 151(3), 1224-1234.

cytes. Immunopharmacol Immunotoxicol (2009). , 38.

moter region. Immunogenetics (1995). , 42, 444-445.

Acad Sci U S A (1998). , 95(16), 9465-70.

Tissue Antigens (1997). , 49, 635-9.

(2000). , 43(1), 120-8.

(2002). , 46(4), 1125-1126.

(1995). , 270, 0618-10624.

120-128.

Opin Immunol (2008). , 20(6), 663-8.

beta knockout mice. J. Immunol (1995).

liferation. J. Immunol (1993). , 150(11), 4754-65.

tion: evidence for a significant association with interleukin-10. Arthritis Rheum (2000). , 43(1), 120-8.

[16] Alfonso, D, Rampi, S, Rolando, M, Giordano, V, & Momigliano-richiardi, M. New polymorphisms in the IL-10 promoter region. Genes Immun 2000;1:231-233., 2000.

transforming growth factor-beta1 production, fibrotic lung disease, and graft fibrosis

[5] Becker-merok, A, Eilertsen, G. Ø, & Nossent, J. C. Levels of transforming growth fac‐ tor-beta are low in systemic lupus erythematosus patients with active disease. J

[6] Bidwell, J, Keen, L, Gallagher, G, Kimberly, R, Huizinga, T, Mcdermott, M. F, Oksen‐ berg, J, Mcnicholl, J, & Pociot, F. Hardt C & D'Alfonso S. Cytokine gene polymor‐ phism in human disease: on-line databases. Genes and immunity (1999). , 1, 3-19. [7] Cambien, F, et al. Polymorphisms of the transforming growth factor-beta 1 gene in relation to myocardial infarction and blood pressure. The Etude Cas-Témoin de l'In‐

[8] Caserta, T. M, Knisley, A. A, Tan, F. K, Arnett, F. C, & Brown, T. L. Genotypic analy‐ sis of the TGF beta-509 allele in patients with systemic lupus erythematosus and

[9] Chen, J. Y, Wang, C. M, Lu, S. C, Chou, Y. H, & Luo, S. F. Association of apoptosisrelated microsatellite polymorphisms on chromosome 1q in Taiwanese systemic lu‐

[10] Chong, W. P, Ip, W. K, Wong, W. H, Lau, C. S, Chan, T. M, & Lau, Y. L. Association of interleukin-10 promoter polymorphisms with systemic lupus erythematosus.

[11] Chung, S. J, Kim, J. S, Jung, H. C, & Song, I. S. Transforming growth factor-[be‐ ta]1-509T reduces risk of colorectal cancer, but not adenoma in Koreans. Cancer Sci

[12] Clancy, R. M, Backer, C. B, Yin, X, Kapur, R. P, Molad, Y, & Buyon, J. P. Cytokine polymorphisms and histologic expression in autopsy studies: contribution of TNF-al‐ pha and TGF-beta 1 to the pathogenesis of autoimmune-associated congenital heart

[13] Crawley, E, Woo, P, & Isenberg, D. A. Single nucleotide polymorphic haplotypes of the interleukin-10 5' flanking region are not associated with renal disease or serology in Caucasian patients with systemic lupus erythematosus. Arthritis and Rheumatism

[14] Alfonso, D, Giordano, S, Mellai, M, Lanceni, M, Barizzone, M, Marchini, N, Scorza, M, Danieli, R, Cappelli, M. G, Rovere, M, Sabbadini, P, & Momigliano-richiardi, M. G. P. Association tests with systemic lupus erythematosus (SLE) of IL10 markers in‐ dicate a direct involvement of a CA repeat in the 5' regulatory region. Genes Immun

[15] Alfonso, D, Rampi, S, Bocchio, M, Colombo, D, Scorza-smeraldi, G, & Momiglianorichardi, R. P. Systemic lupus erythematosus candidate genes in the Italian popula‐

farctus du Myocarde (ECTIM) Study. Hypertension (1996). , 28(5), 881-7.

pus erythematosus patients. Clin Exp Immunol (2006). , 143(2), 281-7.

Sjogren's syndrome. Ann Genet (2004). , 47(4), 359-63.

after lung transplantation. Transplantation (1998). , 66(8), 1014-20.

Rheumatol (2010). , 37(10), 2039-2045.

50 Genes and Autoimmunity - Intracellular Signaling and Microbiome Contribution

Genes Immun (2004). , 5(6), 484-92.

block.J Immunol. (2003). Sep 15;, 171(6), 3253-61.

(2007). , 98(3), 401-4.

(1999). , 42(9), 2017-2018.

(2002). , 3(8), 454-63.

[28] European Bioinformatics InstituteSanger Institute. SNP database: Ensembl Genome Browser. (2006). Available at: http://www.ensembl.org/index.html.

[41] Jin, T, Almehed, K, & Carlsten, H. Forsblad-d'Elia H. Decreased Serum Levels of TGF-β1 are associated with Renal Damages in Female Patients with Systemic Lupus

Gene Polymorphisms of Immunoregulatory Cytokines IL-10 and TGF-β1 in Systemic Lupus Erythematosus

http://dx.doi.org/10.5772/53799

53

[42] Johanneson, B, Lima, G, Von Salomé, J, & Alarcón-segovia, D. Alarcón-Riquelme ME; Collaborative Group on the Genetics of SLE, The BIOMED II Collaboration on the Genetics of SLE and Sjögrens syndrome. A major susceptibility locus for systemic lu‐ pus erythemathosus maps to chromosome1q31. Am J Hum Genet (2002). , 71(5),

[43] Kariuki, S. N, & Niewold, T. B. Genetic regulation of serum cytokines in systemic lu‐

[44] Koss, K, Fanning, G. C, Welsh, K. I, & Jewell, D. P. Interleukin-10 gene promoter polymorphism in English and Polish healthy controls. Polymerase chain reaction haplotyping using 3′ mismatches in forward and reverse primers. Genes and Im‐

[45] Lazarus, M, Hajeer, A. H, Turner, D, Sinnott, P, & Worthington, J. Ollier WER, and Hutchinson IV, Genetic variation in the interleukin 10 gene promoter and systemic

[46] Lebman, D. A, & Edmiston, J. S. The role of TGF-beta in growth, differentiation, and

[47] Li MoWan YY, Flavell RA. T cell-produced transforming growth factor-beta1 con‐ trols T cell tolerance and regulates Th1- and Th17-cell differentiation. Immunity

[48] Li, M. O, Wan, Y. Y, Sanjabi, S, Robertson, A. K, & Flavell, R. A. Transforming growth factor-beta regulation of immune responses. Annu Rev Immunol (2006). , 24,

[49] Lin, Y. J, Wan, L, Huang, C. M, Sheu, J. J, Chen, S. Y, Lin, T. H, Chen, D. Y, Hsueh, K. C, Lai, C. C, Tsai, F. J, & Tnf-alpha, I. L. promoter polymorphisms in susceptibility to systemic lupus erythematosus in Taiwan. Clin Exp Rheumatol (2010). , 28(3), 318-24.

[50] LinS-Y et al., A whole genome methylation analysis of systemic lupus erythemato‐ sus: hypomethylation of the IL10 and IL1R2 promoters is associated with disease ac‐

[51] Liossis, S. N, Kovacs, B, Dennis, G, Kammer, G. M, & Tsokos, G. C. B cells from pa‐ tients with systemic lupus erythematosus display abnormal antigen receptor-mediat‐

[52] Llorente, L, Richaud-patin, Y, & Richaud-patin, Y. Dysregulation of interleukin-10 production in relatives of patients with systemic lupus erythematosus. Arthritis and

ed early signal transduction events. J Clin Invest (1996). , 98(11), 2549-57.

tivity. Genes and Immunity (2012). , 13, 214-220.

Rheumatism (1997). , 40(8), 1429-1435.

lupus erythematosus. Journal of Rheumatology (1997). , 24(12), 2314-2317.

maturation of B lymphocytes. Microbes Infect (1999). , 1(15), 1297-1304.

Erythematosus. Lupus (2011). , 21(3), 310-8.

munity (2000). , 1(5), 321-324.

(2007). May;, 26(5), 579-91.

99-146.

pus erythematosus. Transl Res (2010). , 155(3), 109-17.

1060-71.

[41] Jin, T, Almehed, K, & Carlsten, H. Forsblad-d'Elia H. Decreased Serum Levels of TGF-β1 are associated with Renal Damages in Female Patients with Systemic Lupus Erythematosus. Lupus (2011). , 21(3), 310-8.

[28] European Bioinformatics InstituteSanger Institute. SNP database: Ensembl Genome

[29] Fainaru, O, Shay, T, Hantisteanu, S, Goldenberg, D, Domany, E, & Groner, Y. TGFbe‐ ta-dependent gene expression profile during maturation of dendritic cells. Genes Im‐

[30] Fei, G-Z, Svenungsson, E, Frostegård, J, Padyukov, L, & The, A. IL-10 allele is associ‐ ated with cardiovascular disease in SLE. Atherosclerosis (2004). , 177(2), 409-414.

[31] Fiorentino, D. F, Bond, M. W, & Mosmann, T. R. Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. Jour‐

[32] Flanders, K. C, & Roberts, A. B. TGF-β. In:Ippenheim JJ, Feldmann M (eds). Cytokine

[33] Gateva et alA large-scale replication study identifies TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 as risk loci for systemic lupus erythematosus. Nat Genet