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IDO1 and TGF- 1 β mediate protective effects of IFN-α in antigen-induced arthritis
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
Department of Experimental Medicine, University of Perugia, Perugia, Italy.
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pharmacology.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Interferon-α (IFN-α) prevents antigen-induced arthritis (AIA) in mice by an unknown mechanism. Indoleamine 2, 3 dioxygenase 1 (IDO1) is an immunoregulator via enzymatic as well as signalling activity, which can be activated by TGF-β and further mediated via non canonical NF-κB signalling. We here investigated whether IDO1 and TGF-β are involved in IFN-α protective effects in AIA. Arthritis was induced in wt, Ido1-/- or Ifnar-/- mice, treated or not with IFN-α or kynurenine, the main IDO1 product, and antibodies neutralizing TGF-β or 1-methyltryptophan (1-MT), an inhibitor of IDO1 catalytic activity. IDO1 expression and enzymatic activity were determined by RT-PCR and HPLC, respectively. Proliferation was measured by 3H-Thymidine incorporation. Non-canonical NF-κB signalling was evaluated by ELISA and Western blot in plasmacytoid DCs (pDCs) from treated mice. Protective effects of IFN-α in AIA were associated with increased IDO1 expression and kynurenine production in spleen cells, particularly at the time of mBSA sensitization. Lack of IDO1 ablated IFN-α protection and kynurenine prevented AIA in an IFNAR-independent manner. The IDO1 catalytic activity was crucial for IFN-α effects at the sensitization but not effector phase of AIA. The disease effector phase in mice treated with IFN-α was instead characterized by sustained IDO1 and TGF-β expression and activation of the noncanonical NF-κB pathway in pDCs. IFN-α protective effects in AIA involves IDO1 enzymatic and signalling activity in the disease sensitization and effector phase, respectively. Kynurenine, the main IDO1 metabolite, can be used as an alternative treatment to IFN-α in protecting mice from AIA.

National Category
Pharmacology and Toxicology Rheumatology and Autoimmunity Clinical Medicine
Identifiers
URN: urn:nbn:se:liu:diva-122461OAI: oai:DiVA.org:liu-122461DiVA: diva2:866546
Funder
Swedish Research CouncilSwedish Rheumatism AssociationLinköpings universitet
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2015-11-03Bibliographically approved
In thesis
1. Immune tolerance by interferon-alpha in experimental arthritis
Open this publication in new window or tab >>Immune tolerance by interferon-alpha in experimental arthritis
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type I Interferons (mainly IFN-α & IFN-β) belong to a family of cytokines that possess strong antiviral and immunomodulatory properties. Pro- and/or anti-inflammatory effects of type I IFN have been observed in infectious diseases and several autoimmune diseases including SLE, MS, RA and experimental models thereof, but what defines either outcome is largely obscure. The main aim of this thesis is to understand how IFN-α may act anti-inflammatory in a model of antigen-induced arthritis (AIA). In this model, mice are sensitised with methylated-BSA (mBSA) emulsified in Freund’s adjuvant at day 1 and 7 followed by intra-articular injection of mBSA in the knee joint at day 21, which induces arthritis within 1 week.

Administration of IFN-α at the time of mBSA sensitisations (day 1 and day 7) but not at induction of arthritis (day 21) clearly protected against arthritis in a type I IFN receptor dependent manner. Humoral immunity might not be involved in this protection as the levels of antigen-specific IgG (total, IgG1, IgG2a and IgG2b), IgA, IgE in serum were not altered in IFN-α treated mice. However, IFN-α-protection was accompanied by delayed and decreased antigen-specific proliferative responses in spleen and lymph node cells ex vivo, including impaired proliferative recall responses after intra-articular antigenic challenge.

In the course of AIA, IFN-α inhibited the increase of circulatory IL-6, IL-10, IL-12, and TNF in the sensitisation phase (day 0-21) and also the re-call response of IL-1β, IL-10, IL-12, TNF, IFN-γ, and IL-17 induced by intra-articular mBSA challenge in arthritis phase (day 21-28). This IFN-α-inhibition of cytokines was also apparent in mBSA-re-stimulated spleen and lymph node cell cultures ex vivo, including inhibited cytokine production in CD4+ T helper cells and macrophages. In contrast to the inhibition of pro-inflammatory cytokines, the levels of immunomodulatory TGF-β was clearly enhanced in IFN-α-treated mice, both in serum and in re-stimulated leucocytes cultures including both macrophages, especially in the sensitisation phase, and in CD4+ T cells in the arthritis phase. By  inhibiting TGF-β signalling in vivo, the protective effect of IFN-α was  shown to be dependent on TGF-β signalling in the sensitisation phase.

The cytokine TGF-β is an activator of the indoleamine 2,3 dioxygnese (IDO1), a potent immuneregulatory component that acts via enzymatic production of kynurenine (Kyn) and signalling activity. The IFN-α-protective effect in AIA was associated with both increased expression and enzymatic activity of IDO1 and the IFN-α-protection was totally ablated in mice lacking IDO1 expression (IDO1 KO mice) and in mice treated with the inhibitor of the enzymatic activity of IDO1 (1-Methyl Tryptophan; 1-MT). Interestingly, administration of the IDO-metabolite Kyn protected mice from AIA in an IFNARindependent manner. These observations show that the IDO1 enzymatic activity is important for the protective effect of IFN-α. Using 1-MT, it was further shown that the enzymatic activity of IDO1 was, like TGF-β, crucial only at the sensitisation but not in the arthritis phase of AIA for IFN-α to protect against arthritis. Instead, IDO1’s non-enzymatic signalling activity, characterized by sustained expression of IDO1 and non-canonical NF-κB activation in pDCs, was observed in the arthritis phase in spleen cells from mice treated with IFN-α.

Regulatory T cells (Treg cells) were also found to be important for IFN-α-protection in AIA. Transient depletion of Treg cells by diphtheria toxin in DEREG mice in the arthritis phase, but not during the sensitisation phase abolished IFN-α-protection. Treatment with IFN-α enhanced the numbers of Treg cells in the course of AIA and their function; compared to untreated mice, Treg cells isolated at day 10 and 20 of AIA from IFN-α- treated mice exhibited higher suppressive activity against mBSA-stimulated proliferation of responder T cells. The enhancing effect of IFN-α on Treg cell numbers was observed in blood, spleen, LNs and also in ex-vivo cultures of leucocytes re-stimulated with mBSA and IFN-α. Although IFN-α clearly increased the suppressive activity of Treg cells, adoptive transfer of Treg cells from mBSA immunized mice, regardless of IFN-α treatment, prevented the development of arthritis.

Conclusion

In the presence of IFN-α during antigen sensitisation, a state of tolerance is established, which is able to prevent joint inflammation induced by antigenic re-challenge. This immunological tolerance is created in the sensitisation phase of AIA and is characterized by inhibition of pro-inflammatory cytokines, increased TGF-β production and activity of the IDO1 enzyme, the latter two being indispensable for IFN-α-induced protection. Administration of Kyn, the metabolite of the enzymatic activity of IDO1, in the sensitisation phase also protected against AIA downstream of type I IFN signalling. In the arthritis phase regulatory T cells, whose numbers and suppressive capacity was clearly enhanced by IFN-α, mediate the actual prevention of arthritis development in IFN-α-treated animals. We have thus identified molecular and cellular components of the anti-inflammatory program elicited by IFN-α including Kyn that may not have the pro-inflammatory effects associated with IFN.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. 70 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1495
National Category
Clinical Medicine Pharmacology and Toxicology Rheumatology and Autoimmunity
Identifiers
urn:nbn:se:liu:diva-122463 (URN)10.3384/diss.diva-122463 (DOI)978-91-7685-888-2 (print) (ISBN)
Public defence
2015-12-02, Belladonna, House 511, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research CouncilSwedish Rheumatism AssociationMagnus Bergvall FoundationLinköpings universitet
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2015-11-05Bibliographically approved

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