FICZ and TCDD, two high-affinity AhR ligands, are reported to have opposite effects on T cell differentiation with TCDD inducing regulatory T cells and FICZ inducing Th17 cells. This dichotomy has been attributed to ligand-intrinsic differences in AhR activation, although differences in sensitivity to metabolism complicate the issue. TCDD is resistant to AhR-induced metabolism and produces sustained AhR activation following a single dose in the μg/kg range, whereas FICZ is rapidly metabolized and AhR activation is transient. Nonetheless, prior studies comparing FICZ with TCDD have generally used the same 10-50 μg/kg dose range, and thus the two ligands would not equivalently activate AhR. We hypothesized that high-affinity AhR ligands can promote CD4+ T cell differentiation into both Th17 cells and Tregs, with fate depending on the extent and duration of AhR activation. We compared the immunosuppressive effects of TCDD and FICZ, along with two other rapidly metabolized ligands (ITE and 11-Cl-BBQ) in an acute alloresponse mouse model. The dose and timing of administration of each ligand was optimized for TCDD-equivalent Cyp1a1 induction. When optimized, all of the ligands suppressed the alloresponse in conjunction with the induction of Foxp3- Tr1 cells on day 2 and the expansion of natural Foxp3+ Tregs on day 10. In contrast, a low dose of FICZ induced transient expression of Cyp1a1 and did not induce Tregs or suppress the alloresponse but enhanced IL-17 production. Interestingly, low doses of the other ligands, including TCDD, also increased IL-17 production on day 10. These findings support the conclusion that the dose and the duration of AhR activation by high-affinity AhR ligands are the primary factors driving the fate of T cell differentiation.

Summary Exposure to a plethora of environmental challenges commonly triggers pathological type 2 cell-mediated inflammation. Here we report the pathological role of the Wnt antagonist Dickkopf-1 (Dkk-1) upon allergen challenge or non-healing parasitic infection. The increased circulating amounts of Dkk-1 polarized T cells to T helper 2 (Th2) cells, stimulating a marked simultaneous induction of the transcription factors c-Maf and Gata-3, mediated by the kinases p38 MAPK and SGK-1, resulting in Th2 cell cytokine production. Circulating Dkk-1 was primarily from platelets, and the increase of Dkk-1 resulted in formation of leukocyte-platelet aggregates (LPA) that facilitated leukocytes infiltration to the affected tissue. Functional inhibition of Dkk-1 impaired Th2 cell cytokine production and leukocyte infiltration, protecting mice from house dust mite (HDM)-induced asthma or Leishmania major infection. These results highlight that Dkk-1 from thrombocytes is an important regulator of leukocyte infiltration and polarization of immune responses in pathological type 2 cell-mediated inflammation.

AhR activation by high affinity ligands mediates immunosuppression in association with increased regulatory T cells, making this transcription factor an attractive therapeutic target for autoimmune diseases. We recently discovered 10-chloro-7H-benzimidazo[2,1-a]benzo[de]iso-quinolin-7-one (10-Cl-BBQ), a nanomolar affinity AhR ligand with immunosuppressive activity and favorable pharmacologic properties. Here, we tested the consequences of AhR activation in the NOD model. Oral 10-Cl-BBQ treatment prevented islet infiltration without clinical toxicity, while AhR-deficient NOD mice were not protected. Suppression of insulitis was associated with an increased frequency, but not total number, of Foxp3+ Tregs in the pancreas and pancreatic lymph nodes. The requirement for Foxp3+ cells in AhR-induced suppression of insulitis was tested using NOD.Foxp3DTR mice which show extensive islet infiltration upon treatment with diphtheria toxin. AhR activation prevented the development of insulitis caused by the depletion of Foxp3+ cells, demonstrating that Foxp3+ cells are not required for AhR-mediated suppression and furthermore the AhR pathway is able to compensate for the absence of Foxp3+ Tregs, countering current dogma. Concurrently, the development of disease-associated CD4+Nrp1+Foxp3−RORγt+ cells was inhibited by AhR activation. Taken together, 10-Cl-BBQ is an effective, non-toxic AhR ligand for the intervention of immune-mediated diseases that functions independently of Foxp3+ Tregs to suppress pathogenic T cell development.

Leishmania (Viannia) parasites are etiological agents of cutaneous leishmaniasis in the New World. Infection is characterized by a mixed Th1/Th2 inflammatory response, which contributes to disease pathology. However, the role of T regulatory cells (Treg) in Leishmania (Viannia) disease pathogenesis is unclear. Using the mouse model of chronic L. (V.) panamensis infection, we have examined the hypothesis that Treg functionality contributes to control of pathogenesis. Upon infection, T regulatory cells (CD4+Foxp3+) presented with a dysregulated phenotype, in that they produced IFN-γ, expressed Tbet and had a reduced ability to suppress T cell proliferation in vitro. Targeted ablation of Tregs resulted in enlarged lesions, increased parasite load and enhanced production of IL-17 and IFN-γ with no change in IL-10 and IL-13 levels. Thus, indicating that an increased inflammatory response was commensurate with disease exacerbation and that the remaining impaired Treg cells were important in regulation of disease pathology. Conversely, adoptive transfer of Tregs from naïve mice halted disease progression, lowered parasite burden and reduced cytokine production (IL-10, IL-13, IL-17, IFN-γ). As Tregs appeared important for controlling infection, we hypothesized the expansion of Tregs could be used as an immunotherapeutic treatment approach. As a proof of principle, chronically infected mice were treated with rIL-2-anti-IL-2 antibody complex to expand Tregs. Treatment transitorily increased numbers and percentage of Treg (draining lymph node, spleen), that resulted in reduced cytokine responses, ameliorated lesions and reduced parasite load (105-fold). Thus, immunotherapy targeting Tregs could provide an alternate treatment strategy for leishmaniasis caused by L. (Viannia) parasites.

Activation of the aryl hydrocarbon receptor (AhR) by immunosuppressive ligands promotes the development of regulatory T (Treg) cells. Although AhR-induced Foxp3+ Treg cells have been well studied, much less is known about the development and fate of AhR-induced Type 1 Treg (AhR-Tr1) cells. In the current study, we identified the unique transcriptional and functional changes in murine CD4+ T cells that accompany the differentiation of AhR-Tr1 cells during the CD4+ T-cell-dependent phase of an allospecific cytotoxic T lymphocyte (allo-CTL) response. AhR activation increased the expression of genes involved in T-cell activation, immune regulation and chemotaxis, as well as a global downregulation of genes involved in cell cycling. Increased IL-2 production was responsible for the early AhR-Tr1 activation phenotype previously characterized as CD25+CTLA4+GITR+ on day 2. The AhR-Tr1 phenotype was further defined by the coexpression of the immunoregulatory receptors Lag3 and Tim3 and non-overlapping expression of CCR4 and CCR9. Consistent with the increased expression of CCR9, real-time imaging showed enhanced migration of AhR-Tr1 cells to the lamina propria of the small intestine and colon. The discovery of mucosal imprinting of AhR-Tr1 cells provides an additional mechanism by which therapeutic AhR ligands can control immunopathology.