Groups of control mice were given corn oil alone or BNF at the concentration of 120 mg/kg. Treatment of SU5416 produced a dose-dependent increase in EROD activity in both strains of mice seen after sacrifice (figure 3D), with no significant difference between the two. BNF at 120 mg/kg showed dramatically decreased in vivo activity in the DBA/2J strain. To identify the duration of activity of SU5416 as a ligand of the AHR, we dosed human 101L-hepatoma cells with SU5416 at a dose of 100 nM or TCDD at 1 nM, and measured luciferase activity at 4, 24, 48, 72, and 96 hours. As can be seen in figure S2A, SU5416 has activity at 4 hours, but by 24 hours no longer causes luciferase Table 1. In utero exposure to SU-5416 stimulates closure of DV.
Cos-1 Cells Transfected with AHR Containing Valine Point-mutation Show Similar ED50 to Cos-1 Cells with AHRb Isoform
Because of the importance of identifying that SU5416 is truly unique in its ability to activate the low affinity AHR isoform with similar strength as the high affinity isoform, we transfected COS-1 cells with an AHR containing the same point mutation (valine for alanine) thought to be responsible for the low affinity of the AHRd isoform compared to AHRb. These cells also harbor a luciferase gene next to the DRE. We again tested SU5416 and TCDD, harvesting the cells after 4 hours. As seen in figure 4C, the ED50 for TCDD was higher in the A375V transfected cell line (AHRd type) compared to wild-type, with a much smaller difference between the two isoforms for SU5416 (figure 4D). Specifically, the EC50 for TCDD was 0.73 nM in wild-type cells, and 2.47 nM in transfected cells, while for SU5416 the EC50 was 0.18 nM in wildtype, and 0.31 nM in transfected cells.
Figure 3. In Vitro dose response curves. A. Rat hepatoma cell lines bearing the murine AHRd (AHRd-15) or the rat AHRb (5L) were incubated with increasing doses of TCDD in 96-well plates. After 40 hours, EROD activity was performed from whole cell lysate. (n = 3). B. Same assay as in A, with increasing doses of SU5416. (n = 3). C. Same assay as in A, with increasing doses of BNF. (n = 2). D. Male DBA/2J and C57BL/6J mice (6 weeks old) were orally administered 120 mg/kg BNF, the indicated doses of SU5416 or the vehicle corn oil. Following 48 hours, hepatic microsomal proteins were isolated and used for the assessment of EROD activity. Animals were treated in groups of 4. Error bars: S.D. binding capacity when faced with the valine point mutation see in the AHRd isoform. The data in this figure is normalized from 0 to 100% response. The actual luciferase values are included in figures S3C and D, which again show the potency of these two ligands.
SU5416 Leads to IDO Induction and FoxP3 Upregulation in CD4+ T Cells
An important role for the AHR in the immune system, and specifically T-cell differentiation, has been recognized and continues to be characterized in the literature [6,7]. Some ligands of the AHR have the ability to enhance Treg differentiation from ?naive T-cells (TCDD, kynurenine), while others direct differentiation towards Th17 effector cells (FICZ). We first tested the ability of SU5416 to induce CYP1A1 and CYP1B1 when titrated in solution with cultured splenocytes. Spleens from C57BL/6J mice were harvested and suspended in culture media, and exposed to titrating doses of SU5416. As seen in figure 5A, after 4 hours of culture SU5416 dramatically induced these cytochrome P450 enzymes in a dose-dependent manner, indicating activation of the DRE in vitro. In this same assay we tested the ability of SU5416 to generate the CYP1B1 and the enzyme IDO, the first enzyme in the kynurenine pathway of tryptophan metabolism. IDO has long been known to play a role in Treg generation, and may be central to the mechanism of Dendritic Cell (DC)-directed Treg generation [24]. We as well as others have previously shown that IDO mRNA can be induced by ligands of the AHR, and that the mechanisms of IDO-directed Treg generation may depend on the AHR [25]. This assay shows that SU5416 induced significant amounts of IDO mRNA in splenocytes (figure 5B), a finding that was previously reported for TCDD [26]. To confirm that CYP1A1and IDO induction in splenocytes is in response to an interaction with the AHR and not secondary to an interaction with the VEGF receptor, we compared the response of AHR wild-type and AHR2/2 cells to SU5416 and IFN-c. As shown in figure 5C and D, SU5416 induced CYP1A1 in wild-type but not null cells. Additionally, IDO was induced by SU5416 in wild-type but not null cells, confirming the importance of this receptor in IDO induction. IFN-c did lead to some IDO induction in both wildtype and null cells (although it did not reach statistical significance in the null cells in this representative assay), a finding we have seen in prior experiments [25]. To assess if FoxP3 could be generated by SU5416 exposure, we employed a pDC/T cell coculture. Previous authors have suggested that Treg generation in this assay is driven by IDO production by the plasmacytoid DCs (pDCs) ?[27]. As described in the Methods, naive T-cells were sorted using magnetic bead separation, and placed in culture for 5 days with allogeneic pDCs separated from BALB/C mice. SU5416, TCDD, FICZ, or media alone was added at the start of culture. After 5 days, cells were collected and mRNA harvested for qPCR analysis of IDO and FoxP3. As shown in figure 5E and F, IDO and FoxP3 were generated after addition of SU5416 in this assay. This upregulation was also seen with TCDD, which has been previously reported to induce FoxP3 [6]. In order to look at the ?direct effect of SU5416 on T cells alone, we separated naive CD4+ T cells and exposed them to TGF-b with or without SU516. We used a dose of 2 ng/ml TGF-b, which in our hands has been a suboptimal dose for Treg generation (4 ng/ml has been optimal in our hands). As can be seen in figure 5G, the addition of SU5416 significantly enhanced the FoxP3 protein expression by flow cytometry.
To further support that SU5416 leads to regulatoryFigure 4. Splenocytes from wild-type and AHRd mice analyzed by qPCR for CYP1A1. Spleens from these mice were harvested and suspended in culture media, and exposed to titrating doses of A) TCDD B) SU5416. After 4 hours they were analyzed by qPCR for CYP1A1 analysis. The curves are normalized from 0 to 100% response.
