Systemic and local regulation of experimental arthritis by IFN-α, dendritic cells and uridine

In this thesis, we have studied the immunological processes of joint inflammation that may be targets for future treatment of patients with arthritis. We focus on the immune-modulating properties of interferon-α (IFN-α) and uridine in experimental arthritis. The nucleoside uridine, which is regarded a safe treatment has anti-inflammatory properties notably by inhibiting tumor necrosis factor (TNF) release. Because the inflamed synovium in rheumatoid arthritis (RA) is characterised by pathogenic TNF-production, uridine could potentially be a way to ameliorate arthritis. Systemic administration of uridine had no effect on antigeninduced arthritis (AIA), which is a T-cell dependent model where animals are immunized twice (sensitization) with bovine serum albumin (mBSA), before local triggering of arthritis by intra-articular antigen (mBSA) re-challenge. In contrast, intra-articular administration of uridine clearly down modulated development of AIA in a dose dependent manner and inhibited the expression of synovial adhesion molecules, influx of inflammatory leukocytes and synovial expression of TNF and interleukin 6, but did not affect systemic levels of proinflammatory cytokines or antigen-specific T-cell responses. Local administration of uridine may thus be a viable therapeutic option for treatment of arthritis in the future. Viral double-stranded deoxyribonucleic acid (dsRNA), a common nucleic acid found in most viruses, can be found in the joints of RA patients and local deposition of such viral dsRNA induces arthritis by activating IFN-α. Here we show that arthritis induced by dsRNA can be mediated by IFN-producing dendritic cells in the joint and this may thus explain why viral infections are sometimes associated with arthritis. Earlier, to study the effect of dsRNA and IFN-α in an arthritis model, that like RA, is dependent on adaptive immunity, dsRNA and IFN-α were administered individually during the development of AIA. Both molecules clearly protected against AIA in a type I IFN receptor-dependent manner but were only effective if administered in the sensitization phase of AIA. Here we show that the anti-inflammatory effect of IFN-α is critically dependent on signalling via transforming growth factor β (TGF-β) and the enzymatic activity of indoleamine 2,3 dioxygenase 1 (IDO). The IDO enzyme is produced by plasmacytoid DC and this cell type was critically required both during antigen sensitization and in the arthritis phase of AIA for the protective effect of IFN-α against AIA. In contrast, TGF-β and the enzymatic activity of IDO were only required during sensitization, which indicate that they are involved in initial steps of tolerogenic antigen sensitization. In this scenario, IFN-α first activates the enzymatic activity of IDO in pDC, which converts Tryptophan to Kynurenine, which thereafter activates TGF-β. Common for IDO-expressing pDC, Kyn and TGF-β is their ability to induce development of regulatory T cells (Tregs). We found that Tregs were crucial for IFN-α-mediated protection against AIA, but only in the arthritis phase. In line with this, adoptive transfer of Tregs isolated from IFN-α treated mice to recipient animals in the arthritis phase clearly protected against AIA. The numbers of Tregs were not significantly altered by IFN-α but IFN-α increased the suppressive capacity of Tregs against antigen-induced proliferation. This enhanced suppressive activity of Tregs in the arthritis phase was dependent on the earlier activated enzyme IDO1 during the sensitization phase of AIA. Thus, presence of IFN-α at the time of antigen sensitization activates the enzymatic activity of IDO, which generates Tregs with enhanced suppressive capacity that upon antigen re-challenge prevents inflammation. We have thus identified one example of how immune tolerance can be developed, that may be a future way to combat autoimmunity. CONTENTS Original publications ............................................................................................................................... 1 Sammanfattning....................................................................................................................................... 2 Abbreviations .......................................................................................................................................... 4 Introduction ............................................................................................................................................. 6 Rheumatoid arthritis ........................................................................................................................................... 6 Aetiology ............................................................................................................................................................ 6 Pathogenesis of RA............................................................................................................................................. 7 Role of Cytokines ............................................................................................................................................. 10 Current treatment modalities in RA .................................................................................................................. 11 Tolerogenic approach for treating RA .............................................................................................................. 11 Antigen-Induced arthritis .................................................................................................................................. 12 Type I Interferons ............................................................................................................................................. 12 Type I IFN signalling ................................................................................................................................... 13 Regulation of T cell responses by type I IFNs ............................................................................................. 14 Role of type I IFNs in autoimmunity............................................................................................................ 14 Role of type I IFNs in RA and experimental models of RA ......................................................................... 15 Regulatory T cells ............................................................................................................................................. 16 Effect of type I IFNs on Tregs ...................................................................................................................... 17 TGF-β ............................................................................................................................................................... 17 IDO ................................................................................................................................................................... 18 Modulation of immune system by IDO1 ...................................................................................................... 19 Uridine .............................................................................................................................................................. 20 Aims ...................................................................................................................................................... 21 Experimental design and methods ......................................................................................................... 22 Mice .................................................................................................................................................................. 22 Induction of mBSA-induced arthritis (Paper I, III & IV) ................................................................................. 22 Administration of IFN-α (Paper III, IV) ........................................................................................................... 23 Uridine treatment protocol (Paper I) ................................................................................................................. 23 Other treatment protocols (Paper III) ................................................................................................................ 23 dsRNA induced arthritis (Paper II) ................................................................................................................... 24 Histopathological scoring of arthritis................................................................................................................ 24 Immunohistochemistry (IHC) and scoring (Paper I, II) .................................................................................... 25 Generation of DCs (Paper II) ............................................................................................................................ 26 DC injection protocol (Paper II) ....................................................................................................................... 26 FACS analysis and sorting of DCs (paper II) ................................................................................................... 26 Quantification of Tregs in blood, spleens and LNs (Paper IV) ......................................................................... 27 Ex Vivo assessment of antigen-specific proliferation (Paper I, III) .................................................................. 27 Blood sampling ................................................................................................................................................. 27 ELISA for quantification of anti-mBSA specific IgG (paper I)........................................................................ 28 ELISA for quantification of TGF-β (Paper III) ................................................................................................ 28 ELISA for IFN-α (paper II) .............................................................................................................................. 29 Luminex for quantification of cytokines (paper I) ............................................................................................ 29 Real Time PCR (qPCR) (Paper III) .................................................................................................................. 29 CFSE Suppression assay (Paper IV) ................................................................................................................. 30 In vivo depletion of Tregs (Paper IV) ............................................................................................................... 33 Adoptive transfer of Tregs (Paper IV) .............................................................................................................. 33 Tryptophan and Kynurenine quantification (paper III) ..................................................................................... 34 Results and Discussion .......................................................................................................................... 35 Local but not systemic administration of uridine prevents development of AIA (Paper I) .............................. 35 Systemic administration of uridine does not inhibit development of mBSA-induced arthritis. ................... 35 Local administration of uridine inhibits development of mBSA-induced arthritis in dose dependent manner. ...................................................................................................................................................................... 36 Local administration of uridine suppressed synovial expression of IL-6 & TNF, synovial cell influx and synovial expression of ICAM-1 & CD18 ..................................................................................................... 36 Dendritic Cells activated by double-stranded RNA induce arthritis via autocrine type I IFN signalling (Paper II). ..................................................................................................................................................................... 38 Dendritic cells accumulate in dsRNA-induced arthritis. .............................................................................. 38 dsRNA-activated, Flt3L-derived DCs induce arthritis. ................................................................................ 39 The Arthritogenicity of ds-RNA-activated, Flt3L-derived DCs is dependent on functional IFNAR. .......... 40 Both cDC and pDC are arthritogenic if pretreated with dsRNA. ................................................................. 41 IDO1 and TGF-β Mediate Protective Effects of IFN-α in Antigen-Induced Arthritis (Paper III). ................... 43 TGF-β mediates the protective effect of IFN-α in AIA. ............................................................................... 43 IDO1 mediates the protective effect of IFN-α in AIA. ................................................................................. 44 Kyn, the major IDO1 product, ameliorates arthritis: .................................................................................... 49 The protective effect of IFN-α in AIA requires presence of pDC both in the sensitization and arthritis phase. ............................................................................................................................................................ 50 Regulatory T cells manifest IFN-α mediated protection during antigen-induced arthritis (Paper IV). ............. 52 Regulatory T cells mediate the IFN-α-protection against AIA..................................................................... 52 IFN-α treatment in vivo increases the in vitro suppressive activity of regulatory T cells. ............................ 53 Type I IFN signalling in T helper cells is not required for IFN-α protection against AIA. .......................... 55 Enzymatic IDO activity mediates the increased suppressive capacity of Tregs conferred by IFN-α. .......... 55 Adoptive transfer of Tregs from mBSA immunized mice protects against mBSA-induced arthritis. .......... 57 Summary and Conclusion ..................................................................................................................... 58 Acknowledgement ................................................................................................................................. 60 References ............................................................................................................................................. 62