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. 2009 Jun 15;182(12):7509-17.
doi: 10.4049/jimmunol.0804328.

The immunoregulatory enzyme IDO paradoxically drives B cell-mediated autoimmunity

Grant N Scott  1 , James DuHadaway, Elizabeth Pigott, Natalie Ridge, George C Prendergast, Alexander J Muller, Laura Mandik-Nayak
Affiliations

The immunoregulatory enzyme IDO paradoxically drives B cell-mediated autoimmunity

Grant N Scott et al. J Immunol. .

Abstract

Rheumatoid arthritis (RA) is a chronic and debilitating inflammatory autoimmune disease of unknown etiology. As with a variety of autoimmune disorders, evidence of elevated tryptophan catabolism has been detected in RA patients indicative of activation of the immunomodulatory enzyme IDO. However, the role that IDO plays in the disease process is not well understood. The conceptualization that IDO acts solely to suppress effector T cell activation has led to the general assumption that inhibition of IDO activity should exacerbate autoimmune disorders. Recent results in cancer models, however, suggest a more complex role for IDO as an integral component of the inflammatory microenvironment necessary for supporting tumor outgrowth. This has led us to investigate the involvement of IDO in the pathological inflammation associated with RA. Using the K/BxN murine RA model and IDO inhibitor 1-methyl-tryptophan, we found that inhibiting IDO activity had the unexpected consequence of ameliorating, rather than exacerbating arthritis symptoms. 1-Methyl tryptophan treatment led to decreased autoantibody titers, reduced levels of inflammatory cytokines, and an attenuated disease course. This alleviation of arthritis was not due to an altered T cell response, but rather resulted from a diminished autoreactive B cell response, thus demonstrating a previously unappreciated role for IDO in stimulating B cell responses. Our findings raise the question of how an immunosuppressive enzyme can paradoxically drive autoimmunity. We suggest that IDO is not simply immunosuppressive, but rather plays a more complex role in modulating inflammatory responses, in particular those that are driven by autoreactive B cells.

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Figures

Figure 1
Figure 1. 1MT inhibits arthritis development and inflammatory cytokine production
K/BxN mice were treated with 400mg/kg 1MT b.i.d. (open circles) or carrier alone (filled circles) by oral gavage starting at the age of 21 days. (A) Rear ankles were measured as an indication of arthritis and represented as the (top panel) mean ankle thickness ± SEM and (bottom panel) percentage of mice exhibiting joint inflammation. Representative experiment of 4 total, showing n=5 mice for each treatment. (B) Metatarsal joint from carrier- or 1MT-treated K/BxN mouse at 42d of age stained with H&E. (top panel) scale bar = 500μm, (bottom panel) scale bar = 50μm. Representative sections from a total of n=20 mice for each treatment group. (C) Cells from the joint dLNs (popliteal, axillary, and brachial LNs) were cultured overnight in media alone or PMA (50ng/ml) + Ionomycin (500ng/ml). Cytokines were measured in culture supernatants by cytometric bead array. Graphs show the mean concentration ± SEM from a representative experiment with n=9 carrier-treated (white bars) and 12 1MT-treated (grey bars) mice. This experiment was repeated 3 times. ** p<0.01, * p=0.03
Figure 2
Figure 2. 1MT exerts its effect early in the response
(A) KRN B6.g7 mice were treated with carrier alone (open circles) or 1MT (filled circles) starting at 21d of age and followed for arthritis development. (B) Arthritis was induced in C57BL/6 mice by transfer of arthritic serum on day 0. At the same time, mice began treatment with either 1MT or carrier alone. (C) K/BxN mice were allowed to develop arthritis and then were treated with carrier alone or 1MT b.i.d. starting at the onset of arthritis (33 days of age). Treatment was continued throughout the remainder of the experiment. (D) K/BxN mice were treated with carrier alone or 1MT b.i.d. starting at the age of 21 days. Treatment was stopped 10 days later and the mice followed for arthritis development. Arthritis development was assessed by measurements of rear ankle thickness ± SEM. Representative experiments of 3 total showing n=5 mice for each treatment regimen. ** p<0.01.
Figure 3
Figure 3. IDO is expressed throughout, but has highest activity early in arthritis
(A) IDO protein levels. Joint-draining LNs were harvested from untreated K/BxN mice at the pre/early stage of arthritis (4wk), acute stage of arthritis (6wk), or chronic stage of arthritis (8wk). Total LN protein lysates were immunoprecipitated with affinity-purified rabbit anti-mouse IDO1, separated by SDS-PAGE, and then immunoblotted with monoclonal rat anti-mouse IDO Ig. Epididymis from K/BxN mice and IDO1 deficient mice were used as a positive and negative control, respectively. This is a representative blot of 2 total showing n=2 pre/early and chronic and n=3 acute arthritic LN samples pooled from 2 mice each. (B) Serum kynurenine levels. Kynurenine levels were measured in the serum of individual K/BxN mice at various stages of arthritis. Each symbol represents an individual mouse with a bar depicting the mean. Kynurenine levels are significantly higher at the early arthritic stage than the pre-arthritic (p=0.001), acute arthritic (p=0.0007), and chronic arthritic (p=0.0001) stages.
Figure 4
Figure 4. 1MT treatment does not alter TH1/TH2/TH17 cytokine levels
K/BxN mice were treated with 1MT (grey bars) or carrier alone (white bars) and analyzed at 7–8 wk of age. Cells from the joint dLNs (popliteal, axillary, and brachial LNs) were cultured overnight in media alone or PMA (50ng/ml) + Ionomycin (500ng/ml). Cytokines were measured in culture supernatants by cytometric bead array and/or ELISA. Graphs show the mean concentration ± SEM from a representative experiment with n=9 carrier-treated and 12 1MT-treated mice from a total of 3 separate experiments.
Figure 5
Figure 5. 1MT inhibits the anti-GPI B cell response
K/BxN mice were analyzed at 6–8 wk of age following treatment with either carrier alone, 1MT throughout the course of the experiment (long-term), or 1MT up to the onset of arthritis (short-term). (A) The isotype of anti-GPI Ig was measured by ELISA. IgM, IgG2c, and IgG3 anti-GPI titers were undetectable (data not shown). The data are represented as the mean titer of Ig ± SEM from a total of 6 carrier-treated, 6 long-term 1MT-treated, and 10 short-term 1MT-treated mice. (B) The total amount of serum Ig was measured by ELISA. The data are represented as the mean titer of Ig ± SEM from a total of 6 mice for each treatment group. (C) Antibody Secreting Cells (ASCs) from the spleen, joint draining LN, and non-joint draining-LN were measured using an ELISpot assay. The graph shows the mean number of ASCs ± SEM in each organ from an experiment representative of 3 in total. n=9 carrier-treated 12 long-term and 6 short-term 1MT-treated mice. * p<0.05, ** p<0.02, *** p< 0.01
Figure 6
Figure 6. 1MT does not directly affect B cell activation in vitro
Purified B cells were labeled with CFSE and cultured for 3 days in either media alone, 20 μg/ml anti-IgM (Fab′)2, 2 μg/ml anti-CD40 + 50ng/ml IL-4, or 25μg/ml LPS with or without 100μM 1MT. (A) Proliferation was measured by flow cytometry as a decrease in CFSE intensity. Histograms show plots representative of 3 separate experiments, with histograms from carrier-treated cultures (filled) overlayed with histograms from the corresponding 1MT-treated cultures (open). (B) Total Ig levels were measured in the culture supernatants. Plots show the mean ± SD of triplicate wells from a representative experiment of 3 total.
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