The transcription factor aryl hydrocarbon receptor (AhR) represents a promising therapeutic target in allergy and autoimmunity.
AhR ligands have been shown to be immunosuppressive in a variety of mouse models. AhR signalling induced by the newly described ligand VAF347 inhibits allergic lung inflammation as well as suppresses pancreatic islet allograft rejection. These effects are likely mediated via alterations in dendritic cell (DC) function. Moreover, VAF347 induces tolerogenic DCs. Langerhans cells (LCs) are immediate targets of exogenous AhR ligands at epithelial surfaces; how they respond to AhR ligands remained undefined. Here we asked the question how VAF347 can alter DC function. We used an in vitro model of CD34+ human cord blood hematopoietic progenitor cells that can be differentiated towards different myeloid cell subsets using specific cytokine cocktails.
AhR expression was highly regulated in myeloid subset cells. LCs expressed highest levels of AhR, followed by monocytes. In granulocytes, AhR protein was low to undetectable. Expanded progenitors expressed dim levels of AhR protein. In this study we further demonstrated that AhR agonists such as VAF347 impair LC/DC function by inhibiting their differentiation from monocytic precursors, rather than by interfering with DC maturation. We identified PU.1 as a key molecular target by which AhR agonists control myeloid/DC sub-lineage differentiation. VAF347 addition inhibited PU.1 upregulation in early monocytic cells, thereby resulting in a differentiation inhibition at a CD14 single positive cell state. As above mentioned, the study showed an impairment of DC function upon VAF347 treatment. We were further interested in the effect of VAF347 on LC function when added during differentiation. In these studies we also included the endogenous ligand 6-formylindolo[3,2-b]carbazole (FICZ).
Interestingly, VAF347 and FICZ administration to differentiating cells both led to a decrease in CD1a expression at the end of culture period.
Furthermore, these CD1a+ cells showed a decreased maturation potential.
They had a decreased ability to upregulate activation markers such as CD83 and CD86 compared to control cells. In addition they were not capable of proper cytokine production regarding IL-10 and IL-12p40, and showed a decreased capability to induce a proinflammatory Th1 or Th17 phenotype from naïve T-cells.
We further analysed the endogenous role of AhR during myeloid subset differentiation and maturation. We found that silencing of AhR does not affect monocyte or granulocyte differentiation. However, LC differentiation is slightly impaired upon silencing of AhR. Interestingly, AhR was activated during maturation of cells, and subsequently downregulated. We found p38 induction to be a responsible factor for AhR activation during DC maturation. We therefore suggest that endogenous AhR plays a role during LC maturation, and that its high levels in LCs might render them less responsive to maturation stimuli as compared to DCs or monocytes. In a second project, we established an in vitro culture for human plasmacytoid dendritic cells (pDCs) to further understand differentiation of DC subsets from myeloid precursor cells. For a long time the lymphoid or myeloid origin of these cells remained elusive. We found that human pDC can arise from a myeloid precursor isolated from human cord blood that is positive for CD13 and expresses the myeloid transcription factors (TFs) ICSBP, M-CSF and PU.1 but lacks the erythroid TF GATA-1. We could further show by using retroviral infection experiments that inhibition of RelB together with activation of Notch signalling by the ligand Delta-1 (DL-1) induces pDC differentiation from CD34+ progenitor cells. We therefore suggest the presence of a common human DC progenitor that is directed towards DC or pDC differentiation dependent on the presence of Notch ligands and RelB activation.