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Regulatory T cells manifest IFN-α mediated protection during 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.
Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences.
Region Östergötland, Heart and Medicine Center, Department of Rheumatology.
Twincore, Germany.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Introduction

Type I interferon induces tolerance against arthritogenic antigen and protects against antigen induced arthritis (AIA). Regulatory T cells (Treg cells) resolve aberrant immune reaction, maintain self-tolerance and prevent the development of autoimmune diseases. We here investigated the impact of Interferon alpha (IFN-α) on Treg cells development and function during antigen induced arthritis.

Methods

For AIA, mice were immunized with methylated bovine serum albumin (mBSA) at day 1 and 7 in presence or absence of IFN-α. At day 21, arthritis was induced by intra-articular injection of mBSA and arthritis was evaluated at day 28. At various days of AIA, CD4, CD25, Foxp3 and CTLA-4 expression was quantified by FACS in blood cells, splenocytes, lymph nodes cells and in ex vivo re-stimulated leucocytes (pooled splenocytes and lymph nodes cells) isolated at same days. To investigate the importance of Treg cells in IFN-α protection in AIA, Foxp3DTReGFP+mice were used, where Treg cells can be depleted transiently by administration of diptherin toxin. CFSE based suppression assay was used to assess the suppression by Treg cells isolated day 4, 10, 20 of AIA against proliferation of mBSA or anti-CD3 stimulated responder T cells (Tresp cells) isolated at same days. For adoptive transfer experiments, 250,000 Treg cells from IFN-α treated or untreated mice day 20 of AIA were intravenously injected to recipient pre-immunized mice without IFN-α treatment during the induction of arthritis. The importance of IFN-α signalling on T cells for the IFN-α protection was evaluated by using CD4-Cre+/- IFNAR flox/flox mice.

Results

Protective effects of IFN-α in AIA were associated with significant TGF-β dependent increase in Foxp3+ T cells in blood at day 4 and minor increase of Foxp3+T cells in spleen and lymph node cells. However IFN-α signalling in T cells is not required for IFN-α-protection. Upon ex vivo re-stimulation in presence of IFN-α with mBSA but not anti-CD3, the Treg cells numbers were increased in leucocytes isolated from day 4 and day 10 of AIA. Transient depletion of Treg cells during induction of arthritis (day 21) abolished IFN-α-protection however the protection was not affected when Treg cells are depleted during immunization phase (day 1 and day 7). Against mBSA-stimulated proliferation of Tresp cells, suppression by Treg cells isolated from day 10 and day 20 from IFN-α treated mice are significantly higher than Treg cells from untreated mice. Treg cells isolated from IFN-α or untreated mice at day 20 of AIA when transferred to pre-immunized untreated mice prevent the development of arthritis.

Conclusion

Treg cells are critically associated with IFN-α protective effects in AIA. IFN-α enhances TGF-β dependent early development of Treg cells and later IFN-α enhances their suppressive capacity against T cells proliferation in antigen specific manner during AIA.

Keyword [en]
Interferon alpha, regulatory T cells, antigen induced arthritis, TGF-beta
National Category
Pharmacology and Toxicology Rheumatology and Autoimmunity Clinical Medicine
Identifiers
URN: urn:nbn:se:liu:diva-122462OAI: oai:DiVA.org:liu-122462DiVA: diva2:866560
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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