Proinflammatory properties of IFN-alpha in the joint

Interferon-a is produced in response to viral infections, due to activation of DC by e.g., viral dsRNA. The double strandness of RNA is a common feature of most viruses (Weber, 2006). I recently showed that dsRNA, if delivered directly to a healthy mouse joint, is highly arthritogenic due to its ability to activate production of IFNa (Magnusson, 2006). This innate effect of IFNa may explain why viral infections often result in arthritides and can trigger disease flares in rheumatoid arthritis (RA) (Franssila, 2006). In patients with RA, both myeloid and plasmacytoid DC (the major producers of IFNa) are recruited to the inflamed joints (Jongbloed, 2006). One could therefore hypothezise that dsRNA-triggered IFNa production by synovial DC contribute to arthritis.

Is dsRNA triggered IFN-alpha production by synovial plasmacytoid DC a driving force in Rheumatoid arthritis?

Arthritic symptoms are frequent in patients suffering from various viral infections (Franssila, 2006). I have recently shown that viral dsRNA, is present in the joints of RA patients (Bokarewa, 2008, Magnusson, 2008) and can be arthritogenic due to up-regulation of IFNa production in situ (Magnusson, 2006). It is still unknown which celltype in the joint can produce IFNa in response to dsRNA, and thereby induce arthritis.

In this project, the hypothesis that synovial DC activated to produce IFNa can cause joint inflammation will be tested. Three approaches will be used to test this hypothesis:

First I will determine if DC with known ability to produce IFNa are present in inflamed joints in mice. Arthritis is triggered by intra-articular injection of dsRNA as described (Magnusson, 2006). The analysis of cryosectioned joints will be done with immunohistochemistry using antibodies that can detect plasmacytoid and myeloid DC (the major producers of IFNa).

Second, I will generate DC in vitro, stimulate them with dsRNA, and assay their ability to induce inflammation if deposited in the joint. To generate murine DC, bone marrow cells are matured into DC by the Flt3-ligand as described (Gilliet, 2002). Thereafter the proportion of plasmacytoid and myeloid DC generated cells are analysed by Fluorescence Activated Cell Sorting (FACS) using DC-specific antibodies. To test plasmacytoid and myeloid DC separately, each cell type will be purified using a FACSAria cell sorter. The ability of Flt3L-generated DC (Flt3-DC) to produce IFNa is confirmed by stimulating them with the IFNa-inducer dsRNA. To detect IFNa in cell supernatants I have developed an ELISA (Magnusson, 2006). Generated, sorted DC will be stimulated with dsRNA, thoroughly washed and adoptively transferred intra-articularly to syngeneic mice. Histological evaluation of joints will enable judgement of potential arthritogenic properties of DC stimulated by dsRNA.

Third, I intend to assess the in vivo significance of naturally occurring DC in the development of dsRNA-induced arthritis. To this end both myeloid and plasmacytoid DC will be depleted in vivo by targeting the CD11c-expressing DC as described (Scumpia, 2005). Plasmacytoid DC alone will be depleted using the 120G8 monoclonal antibody (monoclonal rat-anti mouse plasmacytoid DC kindly provided by Schering-Plough) as described (Asselin-Paturel, 2003). Using above procedures, the relative contribution of myeloid DC and plasmacytoid DC in joint inflammation can be determined.

Preliminary results and future experiments

Using immunohistochemistry it was shown that both pDC (120G8+) and CD11c+ DC (mDC) populate the inflamed joints. The plasmacytoid and myeloid DC also are recruited to the inflamed joints of RA patients (Jongbloed, 2006), but their pathogenic role is not known.

To determine if DC may induce inflammation, Flt3L-DCs (35 % myeloid and 40 % plasmacytoid DC as determined by FACS), were stimulated 4h with dsRNA, thoroughly washed, and injected into the knee joints of healthy mice. This treatment of DC led to an inflammatory response in the joints, whereas unstimulated DC did not. This was also the case for sorted populations of plasmacytoid and myeloid DC. Thus both types of DC can mediate dsRNA-induced joint inflammation. To test if the ability to produce IFNa is important for this effect, DC generated from mice unable to produce IFNa will be stimulated with dsRNA and injected in healthy knee joints. Finally, the role of DC in dsRNA-triggered arthritis will be studied by selectively depleting plasmacytoid and myeloid DC before onset of dsRNA-triggered arthritis.

Referenses

• Asselin-Paturel C, Brizard G, Pin JJ, Briere F, Trinchieri G. Mouse strain differences in plasmacytoid dendritic cell frequency and function revealed by a novel monoclonal antibody. J Immunol 2003, 171(12):6466-6477.
• Bokarewa M, Tarkowski A, Lind M, Dahlberg L, Magnusson M. Arthritogenic dsRNA is present in synovial fluid from rheumatoid arthritis patients with an erosive disease course. Eur J Immunol 2008, 38(11):3237-3244.
• Franssila R, Hedman K. Infection and musculoskeletal conditions: Viral causes of arthritis. Best Pract Res Clin Rheumatol 2006, 20(6):1139-1157.
• Gilliet M, Boonstra A, Paturel C et al. The development of murine plasmacytoid dendritic cell precursors is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor. J Exp Med 2002, 195(7):953-958.
• Jongbloed SL, Lebre MC, Fraser AR et al. Enumeration and phenotypical analysis of distinct dendritic cell subsets in psoriatic arthritis and rheumatoid arthritis. Arthritis Res Ther 2006, 8(1):R15.
• Magnusson M, Tarkowski A: Exogenous nucleic acids contribute to joint inflammation in rheumatoid arthritis by activating an anti-viral interferon response. In: Autoimmunity: Role, Regulation and Disorders. Edited by Fynn L. Vogel LFZ: Nova Science Publishers; 2008.
• Magnusson M, Zare F, Tarkowski A. Requirement of type I interferon signaling for arthritis triggered by double-stranded RNA. Arthritis Rheum 2006, 54(1):148-157.
• Scumpia PO, McAuliffe PF, O'Malley KA et al. CD11c+ dendritic cells are required for survival in murine polymicrobial sepsis. J Immunol 2005, 175(5):3282-3286.
• Weber F, Wagner V, Rasmussen SB, Hartmann R, Paludan SR. Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J Virol 2006, 80(10):5059-5064.