The role of IL-27 and IL-35 in inflammatory diseases

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by chronic inflammation within the synovial tissues in multiple joints leading to progressive, erosive destruction of cartilage and underlying joints. The severity of RA is associated with the overexpression of proinflammatory cytokines within the synovial tissue, such as TNF-, IL-1 and IL-6. Recently, IL-17 is thought to play a critical role in maintaining the inflammatory processes within the arthritic joints. Although the etiology and pathogenesis of RA has not been completely elucidated, neutralizing Antibodies against the inflammatory components have been shown to successfully suppress joint inflammation, reduce the relapse rate and delay disease onset in RA patients. Therefore, the expression and regulation of cytokines produced during the disease progression has been the centre of interest in therapeutic studies. Cytokines are important mediators of immune functions in humans and animals. In this thesis, a murine model of RA has been used to investigate the roles of new cytokines Interleukin (IL)-27 and Interleukin (IL)-35. Interleukin (IL)-27, is a heterodimeric cytokine comprised of an IL-12p40 related protein, Epstein-Barr virus-induced gene 3 (EBI3) and a unique IL-12p35 like protein p28. IL-27 is a member of IL-12 family, mainly generated by activated macrophages and dendritic cells. IL-27 binds a receptor composed of WSX-1/TCCR, a ligand-specific chain, and gp130, a signal-transducing molecule shared with other cytokines such as IL-6. IL-27 can promote both pro- and anti-inflammatory immune responses. A novel role of IL-27 regulating autoimmunity has been suggested by experiments on experimental autoimmune encephalomyelitis (EAE) and central nervous system (CNS) inflammation when infected with Toxoplasma gondii. IL-27 suppresses these chronic diseases through inhibiting Th17 activity. Thus, IL-27 may have an important therapeutic potential for treatment of RA in humans. A major aim of this project has been to clone and express a recombinant murine IL-27 in sufficient quantities to study the role of IL-27 in a murine model of RA closely related to the human disease, collagen-induced arthritis (CIA). A short term administration of IL-27 to mice at the onset of the disease had a significantly suppressive effect on disease severity and incidence compared with untreated controls. Mice treated with the recombinant IL-27 also showed reduced serum IL-6, IL-17 and collagen-specific IgG2a. Spleen and lymph node cells from the IL-27-treated mice produced significantly less IFN- and IL-17 compared with cells from the control mice when cultured with collagen in vitro. In contrast, administration of IL-27 to mice during the late phase of CIA significantly exacerbated disease severity. IL-27-treated mice also showed elevated IFN-γ and IL-6 production by the lymphoid cells when compared to untreated mice. However, IL-17 synthesis was not affected between IL-27-treated mice and untreated mice. Consistent with this finding, in vitro IL-27 markedly inhibited the development of Th17 from naïve CD4+, CD4+CD25- and CD4+CD25+ T cells, but had little or no effect on differentiated Th17 cells. Together, these results demonstrated that IL-27 had both pro-inflammatory and anti-inflammatory effects on chronic articular inflammation, mainly associated with Th17 functions. Interleukin (IL)-35, another novel heterodimeric cytokine belonging to IL-12 family, is composed of EBI3 and the IL-12p35 subunit. Little is known about the biological function of IL-35. To study the role of IL-35 in immune responses, murine recombinant IL-35 was cloned and expressed in a mammalian GS system to produce sufficient quantities. IL-35 induced proliferation of murine CD4+CD25+ and CD4+CD25- T cells when stimulated with immobilized anti-CD3 and anti-CD28 antibodies in vitro. The IL-35-expanded CD4+CD25+ T cell population expressed Foxp3 and produced elevated levels of IL-10, whereas the IL-35-induced CD4+CD25- T cells produced IFN-γ but not IL-4. The IL-35-expanded CD4+CD25+ T cells maintained their suppressive functions against CD4+CD25- effector cells. Furthermore, when cultured with soluble anti-CD3 and antigen-presenting cells, IL-35 directly suppressed the proliferation of CD4+CD25- effector cells. Moreover, IL-35 inhibited the differentiation of Th17 cells in vitro. In vivo, IL-35 effectively suppressed established collagen-induced arthritis in mice with the suppression of IL-17 production but enhanced IFN-γ synthesis. Therefore, IL-35 is a novel cytokine suppressing the immune response through the expansion of regulatory T cells and suppression of Th17 cell development. For the future study of human IL-35, human EBI3 and p35 were cloned and linked together with an Fc fusion part. Human IL-35 was expressed in GS system and the function of the recombinant protein needs further study. These data in this thesis provide direct evidence that IL-27 and IL-35 are important mediators in murine collagen-induced arthritis disease. This implicated that IL-27 and IL-35 could represent potential new targets for novel therapeutic agents in human RA. However, the findings on the dual role of IL-27 at the different disease process suggested that the involvement of IL-27 in the pathogenesis of human RA should be carefully investigated before clinical therapy application