CHANGES IN THE LEVEL OF FoxP3+ REGULATORY T LYMPHOCYTES IN PATIENTS WITH EARLY RHEUMATOID ARTHRITIS DURING METHOTREXATE THERAPY

Objective: to analyze the impact of methotrexate (MTX) therapy on percentage and absolute content of FoxP3+ regulatory T lymphocytes (Treg) in the peripheral blood of patients with early rheumatoid arthritis (RA) who had not previously received MTX.

Subjects and methods. The investigation included 45 patients with early RA (2010 ACR/EULAR criteria) who had not previously received MTX, including 39 women; median age was 52.0 [32.5; 57.5] years; disease duration, 5 [4; 6] months, DAS28, 5.01 [4.18; 5.8]; 71.1% of the patients were positive for rheumatoid factor and 88.9% – for anticyclic citrullinated peptide antibodies. As the first disease-modifying antirheumatic drug, all the patients were assigned to receive subcutaneous MTX at an initial dose of 10 mg/week with its rapid escalation up to 20–25 mg/week. The percentage and absolute count of Treg (FoxP3+CD25+; CD152+surface; CD152+intracellular; FoxP3+CD127-; CD25+CD127-; FoxP3+ICOS+; FoxP3+CD154+; and FoxP3+CD274+) were measured by immunofluorescence staining and multicolor flow cytometry.

Results and discussion. At 24 weeks after starting the therapy, median DAS28, SDAI, and CDAI were 3.1 [2.7; 3.62], 7.4 [4.2; 11.4], and 7.0 [4.0; 11.0], respectively; DAS28 and SDAI remission/low disease activity was reached by 22 (56.4%) and 25 (64.1%) patients, respectively; 4 (10.3%) patients had no MTX treatment effect according to the EULAR criteria. After a 6-month course of MTX therapy, the whole group had increases in the percentage of CD4+cells (from 45.0 [38.0; 49.2] to 46.8 [39.9; 53.2]%) and in the percentage and absolute number of CD152+surface from 0.65 [0.22; 1.67] to 2.07 [1.11; 3.81]% and from 0.0002 [0.0001; 0.0008]•109 to 0.0007 [0.0004; 0.002]•109, and a moderate decrease in the percentage and absolute content of FoxP3+ICOS+ cells from 5.3 [2.1; 11.3] to 4.07 [1.6;6.6]% and from 0.002 [0.001-0.006]•109 to 0.0015 [0.0006-0.003]•109 (p<0.05 in all cases).

Conclusion. The use of MTX in early RA is accompanied by an increase in the proportion and number of Treg with a high level of activation markers, which may indicate their enhanced suppressor activity that is more pronounced among the patients who have achieved remission/low disease activity during the treatment.

1. Насонов ЕЛ, Каратеев ДЕ, Балабанова РМ. Ревматоидный артрит. В кн.: Насонов ЕЛ, Насонова ВА, редакторы. Ревматология: Национальное руководство. Москва: ГЭОТАР- Медиа; 2008. С. 290-331 [Nasonov EL, Karateev DE, Balabanova RM. Rheumatoid arthritis. In: Nasonov EL, Nasonova VA, editors. Revmatologiya: Natsional'noe rukovodstvo [Rheumatology: National guidelines]. Moscow: GEOTAR-Media; 2008. P. 290-331].

2. Nielen MM, van Schaardenburg D, Reesink HW, et al. Specific autoantibodies precede the symptoms of rheumatoid arthritis: a study of serial measurements in blood donors. Arthritis Rheum. 2004;50:380-6. doi: 10.1002/art.20018

3. Shi J, van de Stadt LA, Levarht EW, et al. Anti-carbamylated protein (anti-CarP) antibodies precede the onset of rheumatoid arthritis. Ann Rheum Dis. 2014;73:780-3. doi: 10.1136/annrheumdis-2013-204154

4. Kokkonen H, Soderstrom I, Rocklov J, et al. Up-regulation of cytokines and chemokines predates the onset of rheumatoid arthritis. Arthritis Rheum. 2010;62:383-91. doi: 10.1002/art.27186

5. Sakaguchi S. Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses. Ann Rev Immunol. 2004;22:531-62. doi: 10.1146/annurev.immunol.21.120601.141122

6. Miyara M, Gorochov G, Ehrenstein M, et al. Human FoxP3+regulatory T cells in systemic autoimmune diseases. Autoimmun Rev. 2011;10:744-55. doi: 10.1016/j.autrev. 2011.05.004

7. Насонов ЕЛ, Александрова ЕН, Авдеева АС, Рубцов ЮП. Т-регуляторные клетки при ревматоидном артрите. Научно- практическая ревматология. 2014;52(4):430-7 [Nasonov EL, Aleksandrova EN, Avdeeva AS, Rubtsov YP. T-regulatory cells in rheumatoid arthritis. Nauchno-Prakticheskaya Revmatologiya = Rheumatology Science and Practice. 2014;52(4):430-37. (In Russ.)]. doi: 10.14412/1995-4484-2014-430-437

8. Rudensky AY. Regulatory T cells and FoxP3. Immunol Rev. 2011;241:260-8. doi: 10.1111/j.1600-065X.2011.01018.x

9. Cao D, van Vollenhoven R, Klareskog L, et al. CD25+CD4+ regulatory T cells are enriched in inflamed joints of patients with chronic rheumatic disease. Arthritis Res Ther. 2004;6:R335-46. doi: 10.1186/ar1192

10. Jiao Z, Wang W, Jia R, et al. Accumulation of FoxP3- expressing CD4+CD25+ T cells with distinct chemokine receptors in synovial fluid of patients with active rheumatoid arthritis. Scand J Rheumatol. 2007;36:428-33. doi: 10.1080/03009740701482800

11. Sempere-Ortells JM, Perez-Garcia V, Marin-Alberca G, et al. Quantification and phenotype of regulatory T cells in rheumatoid arthritis according to disease activity Score-28. Autoimmunity.2009;42:636-45. doi: 10.3109/08916930903061491

12. Kawashiri S-Y, Kawakami A, Okada A, et al. CD4+CD25(high)CD127(low/-) Treg cell frequency from peripheral blood correlates with disease activity in patients with rheumatoid arthritis. J Rheumatol. 2011;38:2517-21. doi: 10.3899/jrheum.110283

13. Van Amelsfort JMR, Jacobs KMG, Bijlsma JWJ, et al. CD4+CD25+ regulatory T cells in rheumatoid arthritis: differences in the presence, phenotype, and function between peripheral blood and synovial fluid. Arthritis Rheum. 2004;50:2775-85. doi: 10.1002/art.20499

14. Han GM, O’Neil-Andersen NJ, Zurier RB, Lawrence DA. CD4+CD25high T cell numbers are enriched in the peripheral blood of patients with rheumatoid arthritis. Cell Immunol. 2008;253:92-101. doi: 10.1016/j.cellimm.2008.05.007

15. Cao D, Malmstrom V, Baecher-Allan C, et al. Isolation and functional characterization of regulatory CD25brightCD4+ T cells fromthe target organ of patients with rheumatoid arthritis. Eur J Immunol. 2003;33:215-23. doi: 10.1002/immu.200390024

16. Mottonen M, Heikkinen J, Mustonen L, et al. CD4+ CD25+T cells with the phenotypic and functional characteristics of regulatory T cells are enriched in the synovial fluid of patients with rheumatoid arthritis. Clin Exper Immunol. 2005;140:360-7. doi: 10.1111/j.1365-2249.2005.02754.x

17. Liu M-F, Wang C-R, Fung L-L, et al. The presence of cytokine-suppressive CD4+CD25+ T cells in the peripheral blood and synovial fluid of patients with rheumatoid arthritis. Scand J Immunol. 2005;62:312-7. doi: 10.1111/j.1365-3083.2005.01656.x

18. Авдеева АС, Рубцов ЮП, Попкова ТВ и др. Особенности фенотипа Т-регуляторных клеток при раннем ревматоидном артрите. Научно-практическая ревматология. 2016;54(6):660-6 [Avdeeva AS, Rubtsov YP, Popkova TV, et al. Phenotypic features of T regulatory cells in early rheumatoid arthritis. Nauchno- Prakticheskaya Revmatologiya = Rheumatology Science and Practice. 2016;54(6):660-6 (In Russ.)]. doi: 10.14412/1995-4484-2016-660-666

19. Yu X, Wang C, Luo J, et al. Combination with methotrexate and cyclophosphamide attenuated maturation of dendritic cells: inducing Treg skewing and Th17 suppression in vivo. Clin Develop Immunol. 2013;Article ID 238035:12 p.

20. Guggino G, Giardina A, Ferrante A, et al. The in vitro addition of methotrexate and/or methylprednisolone determines peripheral reduction in Th17 and expansion of conventional Treg and of IL-10 producing Th17 lymphocytes in patients with early rheumatoid arthritis. Rheumatol Int. 2015;35:171-5. doi: 10.1007/s00296-014-3030-2

21. Pericolini E, Gabrielli E, Alunno A, et al. Functional improvement of regulatory T cells from rheumatoid arthritis subjects induced by capsular polysaccharide glucuronoxylomannogalactan. PLoS ONE. 2014;9:Article ID e111163.

22. Li Y, Jiang L, Zhang S, et al. Methotrexate attenuates the Th17/IL-17 levels in peripheral blood mononuclear cells from healthy individuals and RA patients. Rheumatol Int. 2012;32:2415-22. doi: 10.1007/s00296-011-1867-1

23. Oh JS, Kim Y-G, Lee SG, et al. Theeffect of various diseasemodifying anti-rheumatic drugs on the suppressive function of CD4+CD25+ regulatory T cells. Rheumatol Int. 2013;33:381-8. doi: 10.1007/s00296-012-2365-9

24. Peres R, Liew F, Talbot J, et al. Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis. PNAS. 2015;122:2509-14. doi: 10.1073/pnas.1424792112

25. Cribbs AP, Kennedy A, Penn H, et al. Regulatory T cell function in rheumatoid arthritis is compromised by CTLA-4 promoter methylation resulting in a failure to activate the IDO pathway. Arthritis Rheum. 2014;66:2344-54. doi: 10.1002/art.38715

26. Cribbs AP, Kennedy A, Penn H, et al. Methotrexate restores regulatory T cell function through demethelation of the FoxP3 upstream enhancer in patients with rheumatoid arthritis. Arthritis Rheum. 2015;67:1182-92. doi: 10.1002/art.39031

27. Kennedy A, Schmiudt EM, Cribbs AP, et al. A novel upstream enhancer of FOXP3, sensitive to methylation-induced silencing, exhibit dysregulatrd methylation in rheumatoid arthritis T reg cells. Eur J Immunol. 2014;44:2668-78. doi: 10.1002/eji.201444453

28. Van Nies JA, Gaujoux-Viala C, Tsonaka R, et al. When does the therapeutic window of opportunity in rheumatoid arthritis close? A study in two early RA cohorts. Ann Rheum Dis. 2014;73 Suppl 2. doi: 10.1136/annrheumdis-2014-eular.5266

29. Cronstein BN. Low-dose methotrexate: A mainstay in the treatment of rheumatoid arthritis. Pharmacol Rev. 2005;57(2):163-72. doi: 10.1124/pr.57.2.3

30. Cronstein BN, Eberle MA, Gruber HE, Levin RI. Methotrexate inhibits neutrophil function by stimulating adenosine release from connective tissue cells. Proc Natl Acad Sci USA. 1991;88(6):2441-5. doi: 10.1073/pnas.88.6.2441

31. Deaglio S, Dwyer KM, Gao W, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med. 2007;204(6):1257-65. doi: 10.1084/jem.20062512

32. Montesinos MC, Yap JS, Desai A, et al. Reversal of the antiinflammatory effects of methotrexate by the nonselective adenosine receptor antagonists theophylline and caffeine: Evidence that the antiinflammatory effects of methotrexate are mediated via multiple adenosine receptors in rat adjuvant arthritis. Arthritis Rheum. 2000;43(3):656-63. doi: 10.1002/1529-0131(200003)43:3<656::AID-ANR23>3.0.CO;2-H

33. Nesher G, Mates M, Zevin S. Effect of caffeine consumption on efficacy of methotrexate in rheumatoid arthritis. Arthritis Rheum. 2003;48(2):571-2. doi: 10.1002/art.10766

34. Montesinos MC, Takedachi M, Thompson LF, et al. The antiinflammatory mechanism of methotrexate depends on extracellular conversion of adenine nucleotides to adenosine by ecto-5’- nucleotidase: findings in a study of ecto-5’-nucleotidase genedeficient mice. Arthritis Rheum. 2007;56(5):1440-5. doi: 10.1002/art.22643

35. Sitkovsky MV, Ohta A. The ‘danger’ sensors that STOP the immune response: The A2 adenosine receptors? Trends Immunol. 2005;26(6):299-304. doi: 10.1016/j.it.2005.04.004

36. Hasko G, Linden J, Cronstein B, Pacher P. Adenosine receptors: therapeutic aspects for inflammatory and immune diseases. Nat Rev Drug Discov. 2008;7(9):759-70. doi: 10.1038/nrd2638

37. Hasko G, Cronstein BN. Adenosine: An endogenous regulator of innate immunity. Trends Immunol. 2004; 25(1):33-9. doi: 10.1016/j.it.2003.11.003

38. Carregaro V, Sa-Nunes A, Cunha TM, et al. Nucleosides from Phlebotomus papatasi salivary gland ameliorate murine collagen- induced arthritis by impairing dendritic cell functions. J Immunol. 2011;187(8):4347-59. doi: 10.4049/jimmunol.1003404

39. Li L, Huang L, Ye H, et al. Dendritic cells tolerized with adenosine A2AR agonist attenuate acute kidney injury. J Clin Invest.2012;122(11):3931-42. doi: 10.1172/JCI63170