Differential Roles of IDO1 and IDO2 in T and B Cell Inflammatory Immune Responses

Abstract

Indoleamine-2,3-dioxygenase (IDO)1 and IDO2 are two closely related tryptophan catabolizing enzymes encoded by linked genes. The IDO pathway is also immunomodulatory, with IDO1 well-characterized as a mediator of tumor immune evasion. Due to its homology with IDO1, IDO2 has been proposed to have a similar immunoregulatory function. Indeed, IDO2, like IDO1, is necessary for the differentiation of regulatory T cells in vitro. However, compared to IDO1, in vivo studies demonstrated a contrasting role for IDO2, with experiments in preclinical models of autoimmune arthritis establishing a proinflammatory role for IDO2 in mediating B and T cell activation driving autoimmune disease. Given their potentially opposing roles in inflammatory responses, interpretation of results obtained using IDO1 or IDO2 single knockout mice could be complicated by the expression of the other enzyme. Here we use IDO1 and IDO2 single and double knockout (dko) mice to define the differential roles of IDO1 and IDO2 in B cell-mediated immune responses. Autoreactive T and B cell responses and severity of joint inflammation were decreased in IDO2 ko, but not IDO1 ko arthritic mice. Dko mice had a reduction in the number of autoantibody secreting cells and severity of arthritis: however, percentages of differentiated T cells and their associated cytokines were not reduced compared to IDO1 ko or wild-type mice. These data suggest that autoreactive B cell responses are mediated by IDO2, while autoreactive T cell responses are indirectly affected by IDO1 expression in the IDO2 ko mice. IDO2 also influenced antibody responses in models of influenza infection and immunization with T cell-independent type II antigens. Taken together, these studies provide evidence for the contrasting roles IDO1 and IDO2 play in immune responses, with IDO1 mediating T cell suppressive effects and IDO2 working directly in B cells as a proinflammatory mediator of B cell responses.

Keywords: B cells; experimental arthritis; immunization; influenza; murine model.

Figure 1

Differential expression of IDO1 and IDO2 in knockout mice. Purified B and T cells were cultured for 48 h either unstimulated (unstim.) or activated (activ.) with LPS + IL-4 (B cells) or anti-CD3 + anti-CD28 (T cells). (A) IDO1 and (B) IDO2 mRNA expression in liver, epididymis, and unstimulated/activated B and T cells isolated from C57BL/6, IDO1 ko, IDO2 ko, and dko mice was measured by qRT-PCR. Symbols represent individual pools of 3 mice and histograms show the mean ± SEM of IDO1 and IDO2 relative to β2M for n = 6 pools/genotype. (C,D) Protein lysates from (C) epididymis, (D) liver, (E) unstimulated/activated B cells, and (F) unstimulated/activated T cells from IDO1 ko, IDO2 ko, dko, or wt C57BL/6 mice were immunoblotted with antibodies to IDO1 and IDO2. Blots were then probed with anti-GAPDH as a loading control. Representative blot of 3 total. Molecular weights are indicated. The IDO2-specific band is indicated with an arrow. Symbols represent individual mice and histograms show the mean ± SEM ratio of (G) IDO1 or (H) IDO2, normalized to GAPDH, relative to the C57BL/6 control for n = 3 blots. (I) Serum and unstimulated/activated B and T cells supernatants (48 h) were analyzed for kynurenine (Kyn) by ELISA. 293-T-REx™ cells stably transfected with murine IDO1, IDO2, or untransfected controls (parental) were used as positive controls for enzyme activity. Symbols respresent individual mice (serum) or pools of 3 mice (B and T cells) and histograms show the mean ± SEM for n = 6/genotype (serum) and n = 3/genotype (B and T cells). P-values were calculated by two-way ANOVA with post-hoc testing by Fisher's LSD test. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, n.s., not significant.

Figure 2

Deletion of both IDO1 and IDO2 inhibits autoantibody production and alleviates arthritis, phenocopying IDO2 single knockout mice. (A) Rear ankles were measured as an indication of arthritis and represented as mean ankle thickness ± SEM from n = 14 wt, n = 10 IDO1 ko, n = 12 IDO2 ko, and n = 9 IDO1/IDO2 (dko) KRN.g7 mice. (B) The number of anti-GPI ASCs from the joint draining lymph node was determined using an ELISpot assay. Symbols represent individual mice and histograms show the mean number of ASC ± SEM from n = 25 wt, n = 21 IDO1 ko, n = 13 IDO2 ko, and n = 18 dko KRN.g7 mice, pooled from a minimum of 4 independent litters of each genotype. P-values were calculated by one way-ANOVA followed by comparison of means with Tukey's post-hoc multiple comparison correction. ^**p < 0.01, n.s., not significant.

Figure 3

Decreased autoreactive T cell activation in IDO2 ko mice is mediated by IDO1. (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). Symbols represent individual mice and histograms show mean % ± SEM of each subpopulation out of total CD4⁺ T cells. (B) Cells from the joint dLNs were cultured for 4 h in PMA + ionomycin + brefeldin A. Intracellular IL-21 was measured by flow cytometry. Symbols represent individual mice and histograms show mean % IL-21⁺ cells ± SEM out of total CD4⁺ T cells. N = 32 wt, n = 21 IDO1 ko, n = 18 IDO2 ko, and n = 18 dko KRN.g7 mice, pooled from 9 independent experiments. (C–E) Cells from the joint dLNs were cultured for 24 h in PMA + Ionomycin. (C) IL-17a, (D) TNFα, and (E) IFNγ were measured in the supernatants by cytometric bead array. Symbols represent individual mice and histograms show mean concentration ± SEM from n = 33 wt, n = 21 IDO1 ko, n = 21 IDO2 ko, and n = 15 dko KRN.g7 mice, pooled from 9 independent experiments. P-values were calculated by one way-ANOVA followed by comparison of means with Tukey's post-hoc multiple comparison correction. ^*p < 0.05, ^**p < 0.01, n.s., not significant.

Figure 4

Antigen presenting function is not affected by deletion of IDO1 or IDO2. Naive KRN T cells were cultured with irradiated splenocytes from wt, IDO1 ko, IDO2 ko, or dko C57BL/6.g7 mice and GPI peptide. (A) Proliferation was measured by the MTS assay at 72 h. Graph is from a representative experiment of 3 total showing mean O.D. ± SD from 3 replicate wells. (B,C) Upregulation of the activation markers CD25 and CD69 were measured by flow cytometry at 48 h. Symbols represent individual mice and histograms show mean fluorescence intensity (MFI) ± SEM from 3 individual experiments. P-values were calculated by one way-ANOVA followed by comparison of means with Tukey's post-hoc multiple comparison correction. ^*p < 0.05, n.s., not significant.

Figure 5

IDO2 does not affect maturation of B cells in the spleen. The frequency of transitional B cell subsets in wt, IDO1 ko, IDO2 ko, and dko spleens were measured by flow cytometry. Transitional B cell subpopulations were defined as T1 (B220⁺CD93⁺IgM^hiCD23⁻), T2 (B220⁺CD93⁺IgM^hiCD23⁺), and T3 (B220⁺CD93⁺IgM^lowCD23⁺). Symbols represent individual mice and histograms show the mean percentage out of total B cells ± SEM from 6 mice per genotype in 2 independent experiments. P-values were calculated by one way-ANOVA followed by comparison of means with Tukey's post-hoc multiple comparison correction. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, n.s., not significant.

Figure 6

IDO2 ko B cells respond normally to stimulation with TLR ligands in vitro. Purified IDO1 ko, IDO2 ko, dko, or wt B cells were labeled with CFSE and stimulated in vitro with Pam3CSK4 (TLR1/2), Poly I:C (TLR3), LPS (TLR4), loxoribine (TLR7), CpG (TLR9), anti-IgM + anti-CD40, PMA + ionomycin, or media alone for 48 h. The cultured cells were then analyzed for (A) proliferation and the upregulation of activation markers (B) CD25, (C) CD80, (D) CD86, and (E) differentiation marker CD138 by flow cytometry. (F) The amount of Ig secreted into the supernatant was measured by ELISA. Symbols represent individual mice and histograms show mean ± SEM, pooled from a minimum of 3 independent experiments. P-values were calculated by one way-ANOVA followed by comparison of means with Tukey's post-hoc multiple comparison correction. n.s., not significant.

Figure 7

IDO2 deficient mice generate reduced antibody response to influenza. Wt, IDO1 ko, IDO2 ko, and dko C57BL/6 mice were infected with 200 TCID₅₀ influenza virus (A/PR/8/34). (A) Titers of anti-PR8 Ig were measured on days 0, 5, 7, and 10 by ELISA. Symbols represent individual mice and histograms show mean serum anti-PR8 Total Ig titer ± SEM for a minimum of n = 10 mice per group. (B) Symbols represent individual mice and histograms show mean serum anti-PR8 IgM vs. IgG titers ± SEM on day 10 from n = 20 wt, n = 15 IDO1 ko, n = 20 IDO2 ko, and n = 20 dko B6 mice. Data are pooled from 4 independent experiments. P-values were calculated by one way-ANOVA followed by comparison of means with Tukey's post-hoc multiple comparison correction. ^**p < 0.01, ^***p < 0.0001, n.s., not significant.

Figure 8

IDO2 ko and dko mice generate reduced antibody responses to T-independent type II, but not T-independent type I or T cell dependent responses. Wt, IDO1 ko, IDO2 ko, and dko C57BL/6 mice were immunized with (A) NP-Ficoll, (B) NP-LPS, or (C) NP-KLH and high affinity anti-NP titers measured by ELISA on day 7 (NP-Ficoll, NP-LPS) or day 10 after the primary and secondary responses (NP-KLH). Symbols represent individual mice and histograms show mean ± SEM of the reciprocal of serum anti-NP IgM and IgG titers from n = 20 mice per group for NP-Ficoll, pooled from 4 independent experiments; n ≥ 15 mice per group for NP-LPS, pooled from 3 independent experiments, and n = 10 mice per group for NP-KLH, pooled from 2 independent experiments. Individual symbols may overlap for mice with identical titers. P-values were calculated using a Mann-Whitney non-parametric test. ^*p < 0.05, ^**p < 0.01, n.s., not significant.

Figure 9

Localization of antibody secreting cells in immunized mice. Wt, IDO1 ko, IDO2 ko, and dko C57BL/6 mice were immunized with (A) NP-Ficoll, (B) NP-LPS, or (C) NP-KLH. Spleens were harvested on day 7 (NP-Ficoll and NP-LPS) or day 10 following primary immunization (NP-KLH). Sections were stained with B220 (red) and Igλ (blue). Representative images are shown from n = 3 mice per genotype from each immunogen. Antibody secreting cells (ASCs) are marked with arrows. Scale bar = 200μm.