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. 2014 Mar 1;192(5):2082-2090.
doi: 10.4049/jimmunol.1303012. Epub 2014 Jan 31.

IDO2 is a critical mediator of autoantibody production and inflammatory pathogenesis in a mouse model of autoimmune arthritis

Lauren M F Merlo #  1 , Elizabeth Pigott #  1 , James B DuHadaway  1 , Samantha Grabler  1 , Richard Metz  2 , George C Prendergast  1   3   4 , Laura Mandik-Nayak  1   5
Affiliations

IDO2 is a critical mediator of autoantibody production and inflammatory pathogenesis in a mouse model of autoimmune arthritis

Lauren M F Merlo et al. J Immunol. .

Abstract

Rheumatoid arthritis and other autoimmune disorders are associated with altered activity of the immunomodulatory enzyme IDO. However, the precise contributions of IDO function to autoimmunity remain unclear. In this article, we examine the effect of two different IDO enzymes, IDO1 and IDO2, on the development of autoimmune arthritis in the KRN preclinical model of rheumatoid arthritis. We find that IDO2, not IDO1, is critical for arthritis development, providing direct evidence of separate in vivo functions for IDO1 and IDO2. Mice null for Ido2 display decreased joint inflammation relative to wild-type mice owing to a reduction in pathogenic autoantibodies and Ab-secreting cells. Notably, IDO2 appears to specifically mediate autoreactive responses, but not normal B cell responses, as total serum Ig levels are not altered and IDO2 knockout mice are able to mount productive Ab responses to model Ags in vitro and in vivo. Reciprocal adoptive transfer studies confirm that autoantibody production and arthritis are modulated by IDO2 expression in a cell type extrinsic to the T cell. Taken together, our results, provide important insights into IDO2 function by defining its pathogenic contributions to autoantibody-mediated autoimmunity.

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Figures

Figure 1
Figure 1. Deletion of IDO2, but not IDO1 ameliorates arthritis
(A) Rear ankles were measured as an indication of arthritis and represented as the mean change in ankle thickness ± SEM from n=14 KRN.g7, n=10 IDO1 ko KRN.g7, and n=12 IDO2 ko KRN.g7 mice, pooled from 3 independent litters per genotype. (B) Metatarsal joint from KRN.g7, IDO1 ko KRN.g7, and IDO2 ko KRN.g7 mice at 42d of age stained with H&E. Representative sections from a total of n=14 KRN.g7, n=10 IDO1 ko KRN.g7, and n=12 IDO2 ko KRN.g7 mice. Scale bar = 100μm. *p<0.05. n.s., not significant.
Figure 2
Figure 2. IDO expression, kynurenine production, and the effect of the IDO inhibitor 1MT in IDO1 ko and IDO2 ko KRN.g7 mice
(A) IDO2 and (B) IDO1 mRNA expression was measured by real-time PCR. Data show the median and interquartile range of the fold change in IDO expression relative to KRN.g7 control mice. (A) KRN.g7: n=13 (spleen and liver), IDO1 ko KRN.g7: n=8 (spleen and liver); (B) KRN.g7: n=11 (spleen and liver), IDO2 ko KRN.g7: n=7 (spleen), n=6 (liver). (C) Serum kynurenine levels were measured by mass spectroscopy. Data show mean ± SEM, n=8 mice per group, pooled from 3 independent litters of each genotype. (D) KRN.g7, IDO2 ko KRN.g7, and IDO1 ko KRN.g7 mice were treated with 400mg/kg D/L 1-MT or carrier alone beginning at 21 days of age. Data show mean change in ankle thickness ± SEM from n=10 1MT-treated and n=7 Carrier-treated KRN.g7, n=5 1MT-treated and n=5 Carrier-treated IDO1 ko KRN.g7 and IDO2 ko KRN.g7 mice, pooled from 2 independent experiments. *p<0.05. n.s., not significant.
Figure 3
Figure 3. IDO2 affects anti-GPI B cell response but not total antibody levels
(A) Anti-GPI Ig titer in serum from KRN.g7, IDO1 ko KRN.g7, and IDO2 ko KRN.g7 mice was determined by ELISA. Data are represented as mean titer of Ig ± SEM, n=8 mice per group, pooled from 3 independent litters of each genotype. (B) The number of anti-GPI ASCs from joint dLN was determined using an ELISPOT assay. Data shows the mean number of ASC ± SEM for n=27 KRN.g7, n=11 IDO1 ko KRN.g7, and n=12 IDO2 ko KRN.g7 mice, pooled from 3 independent litters of each genotype. (C) Total serum Ig level was measured by ELISA. Mean Ig ± SEM is shown for n=12 mice/group, pooled from 3 independent litters of each genotype. *p<0.05. n.s., not significant.
Figure 4
Figure 4. IDO2 deficient mice develop arthritis in response to transferred arthritogenic antibodies
Arthritis was induced in wt and IDO2 ko C57BL/6 mice by transfer of arthritic serum on day 0. Data show mean change in ankle thickness ± SEM for n=12 wt and n=9 IDO2 ko mice, pooled from 2 independent experiments. n.s., not significant.
Figure 5
Figure 5. IDO2 does not affect B cell response to model antigens
Purified B cells, pooled from 2 each of C57BL/6 or IDO2 ko C57BL/6 mice, were labeled with CFSE and cultured for 3 days in either media alone, 25μg/ml LPS, or 2μg/ml anti-CD40 + 50ng/ml IL-4. (A) Proliferation was measured by flow cytometry as a decrease in CFSE intensity. Graphs show mean ± SEM of percent proliferated cells, gated on B220+ cells, pooled from 3 independent experiments. (B) Total Ig levels were measured in the culture supernatants. Plots show the mean ± SEM, pooled from 3 independent experiments. (C) wt and IDO2 ko C57BL/6 mice were immunized with NP-KLH precipitated in alum and high affinity anti-NP Ig titer measured by ELISA 8 days after initial immunization to measure primary response and again 8 days following a second immunization to quantify secondary response. Graph shows mean ± SEM of the reciprocal of serum anti-NP titer from n=10 mice per group, pooled from 2 independent experiments. n.s., not significant.
Figure 6
Figure 6. IDO2 alters helper T cell populations and cytokine levels
(A) Frequency of CD4+ T helper cell subpopulations were measured by flow cytometry by intracellular staining for the transcription factors T-bet (Th1), GATA-3 (Th2), RORγt (Th17), Bcl-6 (Tfh), and FoxP3 (Treg). Graphs show mean % ± SEM of each subpopulation out of total CD4+ T cells for n=14 IDO2 ko KRN.g7 and n=13 KRN.g7 mice, pooled from 3 independent experiments. (B) Cells from the joint dLNs (popliteal, axillary, and brachial LNs) were cultured overnight in PMA (50ng/ml) + ionomycin (500ng/ml). Cytokines were measured in culture supernatants by cytometric bead array. Graphs show the mean concentration ± SEM from n=12 IDO2 ko KRN.g7 and n=22 KRN.g7 mice (except n=21 for MCP-1 and RANTES), pooled from 3 independent experiments. (C) Cells from the joint dLNs were cultured for 4h in PMA (50ng/ml) + ionomycin (500ng/ml) + 3μg/ml brefeldin A. Intracellular IL-21 was measured by flow cytometry. Graph shows % IL-21+ cells ± SEM out of total CD4+ and CD8+ T cell populations from n=12 IDO2 ko KRN.g7 and n=15 KRN.g7 mice, pooled from 4 independent experiments. *p<0.05. n.s., not significant.
Figure 7
Figure 7. Effect of IDO2 is not intrinsic to T cells
Arthritis was induced by the adoptive transfer of purified CD4+ KRN T cells from wt or IDO2 ko C57BL/6 (I-Ab) mice into T-cell deficient wt or IDO2 ko C57BL/6 (I-Ag7/b) hosts. (A) Schematic for reciprocal adoptive transfer strategy. (B) Rear ankles were measured as an indication of arthritis and represented as the mean change in ankle thickness ± SEM. (C) Serum anti-GPI titers were measured in recipient mice 14 days after T cell transfer. Data shows the mean ± SEM of the reciprocal of serum anti-GPI titer. Data is from n=14 wt into wt, n=12 IDO2 ko into wt, n=6 wt into IDO2 ko, and n=7 IDO2 ko into IDO2 ko mice, pooled from 5 independent experiments. *p<0.05, **p<0.01. n.s., not significant.
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