Figure 1.
TCDD and FICZ administration inhibit EAE establishment and dampen T cell expansion and infiltration of the central nervous system.
A) Clinical scores of mice immunized with MOG/CFA which received either vehicle (filled circles), TCDD (inverted triangles) or FICZ (filled squares) i.p. at the time of immunization; table showing incidence, mean day of onset and mean maximum score of disease. Data shown as mean ± SEM are representative of 2 independent experiments, ***p<0.001, **p<0.01 in two-way ANOVA followed by Dunnet post-test. B) Number of IL-17A+, and number and frequency of Foxp3+ T cells recovered from draining lymph nodes on day 6 post immunization in mice that received TCDD. Data shown as mean ± SEM are representative of 2 independent experiments. *p<0.05 in Student’s T test. C) Number of IL-17A+, IFN-γ+ and Foxp3+ T cells, and frequency of Foxp3+ T cells recovered from the spinal cord on day 15 post immunization in mice that received TCDD. Data shown as mean ± SEM are representative of 2 independent experiments. *p<0.05 in Student’s T test. D) Number of IL-17A+, and number and frequency of Foxp3+ T cells recovered from draining lymph nodes on day 6 post immunization in the presence of FICZ. E) Number of IL-17A+, IFN-γ+ and Foxp3+ T cells, and frequency of Foxp3+ T cells recovered from the spinal cord on day 15 post immunization in mice that received FICZ. Data shown as mean ± SEM are representative of 2 independent experiments.
Figure 2.
Administration of TCDD after initiation of the immune response does not result in immunosuppression.
A) Clinical scores of mice immunized with MOG/CFA which received either vehicle (filled circles) or TCDD i.p. at the time of immunization (open circles) or 7 days after immunization (open squares). Table showing incidence, mean day of onset and mean maximum score of disease. Data shown as mean ± SEM are representative of 2 experiments, **** p<0.0001, two-way ANOVA followed by Dunnet post-test. B) Number of Foxp3+ and of IL-17A+ T cells recovered at day 9 after immunization from draining lymph nodes of immunized mice who received vehicle, TCDD on day 0 or TCDD on day 7 after immunization; n = 4. Data shown as mean ± SEM are representative of 2 experiments, *p<0.05, **p<0.01 in one-way ANOVA followed by Dunnet post-test.
Figure 3.
AhR activation by TCDD and FICZ in vitro promotes Th17 but not Foxp3+ Treg cells.
A) Left panel: ahr expression from in vitro differentiated Th0, Th17 and Treg cells, and ex vivo sorted natural Th17 (nTh17) and Treg cells (nTreg) Data shown as mean ± SEM are representative of 2 experiments; n = 2–4. Right panel: cyp1a1 expression from in vitro differentiated Th0 or Th17 in the presence or absence of FICZ. Data shown as mean ± SEM are representative of 2 experiments; n = 3. B) Fold increase of foxp3 expression in cultured naïve T cells activated in the presence of TCDD, FICZ or TGFβ over control conditions without mediators at the indicated timepoint. C) Representative FACS dot plots showing Foxp3 staining of sorted naïve T cells activated in the presence of indicated ligands and cytokines. D) Representative FACS dot plots showing IL-17A and IL-22 staining in naïve T cell activated in the presence of IL-6 and TGFβ, plus the depicted AhR ligands. E) Representative histogram showing proliferation of in vitro differentiated Th17 cells (gated on IL-17+ cells) in the presence or absence of TCDD by CTV dilution; table shows the average division index (average number of cell divisions each cell has undergone), replication index (fold-expansion of dividing cells) and percentage of divided cells for each group, and the corresponding p value from Student’s T test analysis. F) Representative FACS dot plots showing IL-17A and Foxp3 staining in naïve T cell co-cultured with bone marrow derived DC in the presence of the indicated cytokines and ligands. Data are representative of 2 independent experiments.
Figure 4.
AhR positively controls IL-1 receptor type 1 on Th17 cells.
A) Naïve CD4+ T cells from WT B6 (black bars) and AhR−/− mice (white bars) were differentiated into Th0, Th1, Th2, iTreg, and Th17 cells for 3 days and the relative gene expression of Il-1r1 was determined by quantitative RT-PCR. B) Il-1r1 expression of WT Th17 cells cultured with (filled circles) or without FICZ (open circles) and AhR−/− Th17 cells (open squares) was assessed at 18, 24, and 48h after the onset of Th17 differentiation. C) Th17 cells were induced from naïve WT CD4+ T or AhR−/− naïve CD4+ T cells for 3 days and the expression level of surface IL-1R1 was analyzed gated on IL-17-producing cells. Data are representative of at least two independent experiments.
Figure 5.
ahrb1 and ahrd carrying mice show similar in vitro Th17 differentiation and in vivo disease kinetics following MOG-CFA immunization.
A) Frequency of IL-17A (left panel) and IL-22 (right panel) -expressing cells following culture of naïve CD4+ T cells in Th17-promoting conditions in the presence of different AhR ligands for 4 days; cells bear either the ahrb1 (black bars) or the ahrd (grey bars) allele for AhR. n = 3–4, data shown as mean ± SEM are representative of 2 independent experiments. B) mRNA expression of il17a (left panel) and il22 (right panel) in Th17 cells cultured in the presence of different AhR ligands for 4 days; cells bear either the ahrb1 (black bars) or the ahrd (grey bars) allele for AhR. n = 3–4, data shown as mean ± SEM are representative of 2 independent experiments. *** p<0.001 in a two-way ANOVA test. C) Quantification of IL-17A (left panel) and IL-22 (right panel) protein levels in supernatant obtained from Th17 cells cultured in the presence of different AhR ligands for 4 days; cells bear either the ahrb1 (black bars) or the ahrd (grey bars) allele for AhR. n = 3–5, data shown as mean ± SEM are representative of 2 independent experiments. *** p<0.001 in a two-way ANOVA test. D) Clinical score of mice bearing either ahrb1 (closed circles) or the ahrd (open circles) allele for AhR, immunized with CFA emulsified with MOG peptide. n = 8 mice per group.
Figure 6.
Restricting AhR deficiency to either T cells or dendritic cells partially abrogates TCDD mediated suppression of EAE.
A) ahr mRNA expression in FACS sorted CD4 T cells from AhRfl/+ or AhRfl/− Rag1.Cre mice. B) Clinical score of mice with Rag1-specific AhR-deletion (open circles, open triangles) or control mice immunized with MOG/CFA (filled circles, filled triangles) which received either vehicle (circles) or TCDD (triangles) i.p. at the time of immunization. n = 6–8, ** p<0.01 in two-way ANOVA followed by Dunnet post-test. C) Table showing incidence, mean day of onset and mean maximum score of disease for both experimental groups; n = 6-8, data shown as mean ± SEM. D) ahr expression in FACS sorted CD11c+ MHCII+ cells from AhRfl/+ or AhRfl/− CD11c.Cre mice. E) Clinical score of mice with CD11c-specific AhR-deletion (open circles or open triangles) or control mice (filled circles, filled triangles) immunized with MOG/CFA which received either vehicle (circles) or TCDD (triangles) i.p. at the time of immunization. F) Table showing incidence, mean day of onset and mean maximum score of disease for both experimental groups; n = 9–11, data shown as mean ± SEM, * p<0.05 in two-way ANOVA followed by Dunnet post-test.