Loading...
 
Back to the list
Mediterr J Rheumatol 2016;27(3):119-20
Article Links
The role of regulatory B cells in the homeostasis of regulatory T cells and Th17 cells in patients with systemic sclerosis
Athanasios Mavropoulos, George Efthymiou, Dimitrios P. Bogdanos, Lazaros I. Sakkas
Authors Information
Department of Rheumatology and Clinical Immunology, Faculty of Medicine, School of Health Sciences, University of Thessaly and University General Hospital of Larissa, Thessaly, Greece
Abstract

This paper has no Abstract

Keywords: Tregs, Bregs, Th17, systemic sclerosis
Full Text

BACKGROUND

Systemic sclerosis (SSc) is characterized by fibrosis, activation of the immune system and microvasculopathy.1-8 In recent years, regulatory B cells (Bregs) have been shown to play an important role in the development of autoimmune diseases. The breakdown of immunological tolerance is characterized by the predominance of autoreactive B cells and the lack of Bregs.9-10

Bregs include CD19 (+) CD24 (high) CD38 (high) (transitional Bregs) and memory Bregs. These cells suppress the activity of other cells mainly through the induction of interleukin-10 (IL-10).11 The role of Bregs has been investigated in murine models of experimental autoimmune diseases and human studies of various autoimmune diseases. In SSc there is a disturbance of homeostasis of B cells with evidence of hyperactivation of B cells producing pathogenic autoantibodies.12 Our research group has recently showed insufficiency of Bregs in patients with SScl.13 In particular, both transitional Bregs and memory Bregs are numerically decreased, more profoundly in diffuse than limited cutaneous form of the disease. Interestingly, patients with SSc-associated pulmonary fibrosis have near total lack of transitional and memory Bregs.13 Furthermore, there is functional impairment of Bregs (inability to produce IL-10)13

The anti-inflammatory/suppressory activity of IL-10 and the immunomodulatory action on B and T autoreactive cell populations has been documented.14 Recent studies have highlighted the key role played by Bregs in the fine balance between regulatory T cells (Tregs) and Th17 cells.15-17 It appears that in vitro Bregs induce T lymphocytes to express Tregs cell markers (CD25, FoxP3); leading them to produce IL-10.18-19 It seems that the reverse is also true, since Tregs induce the production of Bregs in vitro.20

In some of our patients, we observed subpopulations of B cells which could produce small, but not negligible, IL-10. This leads us to the hypothesis that these populations may be potential reservoirs of immunoregulatory B lymphocytes. These cells were detectable even in patients with advanced diffuse SSc. We will isolate these subpopulations of B lymphocytes and attempt to differentiate them in inducible Bregs analogously with inducible Tregs.

Aims and Scope

The purpose of the study is twofold: after characterizing the cell subpopulations of Bregs, Tregs and Th17 in patients with SScl, we will study the ability of Bregs to induce Tregs in vitro.

In particular, the aims of the current proposal are: a) to assess Bregs (transitional and memory), Tregs and Th17 in patients with SSc; b) in vitro to induce Bregs; c) to study in vitro the effect of Bregs on Tregs (and vice versa) in patients with SSc.

Material and Methods

We will study Bregs, Tregs and Th17 from patients with SSc (early disease and established disease, limited or diffuse cutaneous form of the disease), and also from patients with rheumatoid arthritis-associated pulmonary fibrosis as disease controls, and healthy subjects as normal controls. The research protocol will be performed in accordance to the revised Helsinki declaration and is approved by the Ethical Committee of the University General Hospital of Larissa, University of Thessaly. Patients undergo regular blood tests during follow up according to good clinical practice, and an additional 15-20 ml of blood sample is taken once for the purpose of this study. PBMCs are kept in liquid nitrogen until used. Isolated peripheral blood mononuclear cells (PBMCs) from patients and controls will be stored in liquid nitrogen and are readily available from the Biobanking Facility of the Department of Rheumatology and Clinical Immunology, University of Thessaly.

Phenotypic characterization of B cell subpopulations will be performed by standard flow cytometry protocols using fluorochrome-conjugated monoclonal antibodies against surface markers such as CD19, CD24, CD38, and CD27. Negative selection based on magnetic beads will allow isolation of B or T cells to be used for the induction of regulatory subsets using non-specific and specific stimuli (polyclonal IgM, BCR activation; bacterial CpG, ODN2006:TLR-9 activation; bacterial LPS: TLR-4 activation), PMA and ionomycin which will be used in order to maximise induction of IL-10. Standard intracellular flow cytometric analysis will be used to measure cytokine expression. Cell cultures in the presence of immunomodulatory agents will assist the induction of Bregs and Tregs in addition to physiological agents, such as IL-2 and ΙL-15.

Tregs will be identified by staining for cell surface CD25 and transcriptional factor FoxP3. B cells will be cultured with autologous CD4(+) T cells and the effect will be assessed by proliferation assays and/or cytokine production, such as IFNγ and IL-6.

Importance of the Study - Anticipated Results

We anticipate that our study will obtain data that could elucidate the pathogenic mechanisms responsible for the development of SSc. The ultimate objective of the study is to find the most effective therapeutic strategy for SSc. This therapy will be based on autologous transfusion of inducible Bregs (alone or in combination with inducible Tregs).To this end, we will first see whether B lymphocytes in patients with SSc have the potential to convert into Bregs and to produce IL-10. Our research group is in an advantageous position with regard to the study of Bregs and their interaction with the Treg/Th17, since we have thoroughly studied these cells and were the first group to show the important role of Bregs in SSc.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.
References
  1. Sakkas L I, Bogdanos D P. Systemic sclerosis: New evidence re-enforces the role of B cells. Autoimmun Rev 2016;15:155-61.
  2. Sakkas L I, Simopoulou T, Katsiari C, Bogdanos D, Chikanza I C. Early systemic sclerosis-opportunities for treatment. Clin Rheumatol 2015;34:1327-31.
  3. Cantarini L, Rigante D, Vitale A, Napodano S, Sakkas L I, et al. Intravenous immunoglobulins (IVIG) in systemic sclerosis: a challenging yet promising future. Immunol Res 2015;61:326-37.
  4. Sakkas L I, Chikanza I C, Platsoucas  . Mechanisms of Disease: the role of immune cells in the pathogenesis of systemic sclerosis. Nat Clin Pract Rheumatol 2006;2:679-85.
  5. Sakkas L I. New developments in the pathogenesis of systemic sclerosis. Autoimmunity 38:113-6.
  6. Kalogerou A, Gelou E, Mountantonakis S, Settas L, Zafiriou E, et al. Early T cell activation in the skin from patients with systemic sclerosis. Ann Rheum Dis 2005;64:1233-5.
  7. Sakkas L I, Platsoucas C D. Is systemic sclerosis an antigen-driven T cell disease? Arthritis Rheum 2004;50: 1721-33.
  8. Sakkas L I, Xu B, Artlett C M, Lu S, Jimenez S A, et al. Oligoclonal T cell expansion in the skin of patients with systemic sclerosis. J Immunol 2002;168:3649-59.
  9. Yoshizaki A, Miyagaki T, DiLillo D J, Matsushita T, Horikawa M, et al. Regulatory B cells control T-cell autoimmunity through IL-21-dependent cognate interactions. Nature 2012;491:264-8.
  10. Xing C, Ma N, Xiao H, Wang X, Zheng M, et al. Critical role for thymic CD19+CD5+CD1dhiIL-10+ regulatory B cells in immune homeostasis. J Leukoc Biol 2015;97:547-56.
  11. Khoder A, Sarvaria A, Alsuliman A, Chew C, Sekine T, et al. Regulatory B cells are enriched within the IgM memory and transitional subsets in healthy donors but are deficient in chronic GVHD. Blood 2014;124:2034-45.
  12. Sato S, Fujimoto M, Hasegawa M, Takehara K. Altered blood B lymphocyte homeostasis in systemic sclerosis: expanded naive B cells and diminished but activated memory B cells. Arthritis Rheum 2004;50:1918-27.
  13. Mavropoulos A, Simopoulou T, Varna A, Liaskos C, Katsiari C, et al. B regulatory cells are decreased and functionally impaired in patients with systemic sclerosis. Arthritis Rheumatol.2016;68:494-504.
  14. Lampropoulou V, Hoehlig K, Roch T, Neves P, Calderon Gomez E, et al. TLR-activated B cells suppress T cell-mediated autoimmunity. J Immunol 2008;180:4763-73.
  15. Carter N A, Rosser E C, Mauri C. Interleukin-10 produced by B cells is crucial for the suppression of Th17/Th1 responses, induction of T regulatory type 1 cells and reduction of collagen-induced arthritis. Arthritis Res Ther 2012;14:R32.
  16. Flores-Borja F, Bosma A, Ng D, Reddy V, Ehrenstein M R, et al. CD19+CD24hiCD38hi B cells maintain regulatory T cells while limiting TH1 and TH17 differentiation. Sci Transl Med 2013;5:173ra123.
  17. Kim H Y, Cho M L, Jhun J Y, Byun J K, Kim E K, et al. The imbalance of T helper 17/regulatory T cells and memory B cells during the early post-transplantation period in peripheral blood of living donor liver transplantation recipients under calcineurin inhibitor-based immunosuppression. Immunology 2013;138:124-33.
  18. Shah S, Qiao L. Resting B cells expand a CD4+CD25+Foxp3+ Treg population via TGF-beta3. Eur J Immunol 2008;38:2488-98.
  19. Kessel A, Haj T, Peri R, Snir A, Melamed D, et al. Human CD19(+)CD25(high) B regulatory cells suppress proliferation of CD4(+) T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells. Autoimmun Rev 2012;11:670-7.
  20. Zheng M, Xing C, Xiao H, Ma N, Wang X, et al. Interaction of CD5 and CD72 is involved in regulatory T and B cell homeostasis. Immunol Invest 2014;43:705-16.