Reciprocal role of GATA-1 and vitamin D receptor in human myeloid dendritic cell differentiation.

Two major pathways of human myeloid dendritic cell (DC) subset differentiation have previously been delineated. Langerhans cells (LCs) reside in epithelia in the steady state, whereas monocytes can provide dendritic cells (DCs) on demand in response to inflammatory signals. Both DC subset pathways arise from shared CD14+ monocyte precursors, which in turn develop from myeloid committed progenitor cells. However, the underlying hematopoietic mechanisms still remain poorly defined. Here, we demonstrate that the vitamin D(3) receptor (VDR) is induced by transforming growth factor beta1 during LC lineage commitment and exerts a positive role during LC generation. In contrast, VDR is repressed during interleukin-4 (IL-4)-dependent monocyte-derived DC (moDC) differentiation. We identified GATA-1 as a repressor of VDR. GATA-1 is induced by IL-4 in moDCs. Forced inducible expression of GATA-1 mimics IL-4 in redirecting moDC differentiation and vice versa, GATA-1 knockdown arrests moDC differentiation at the monocyte stage. Moreover, ectopic GATA-1 expression stabilizes the moDC phenotype under monocyte-promoting conditions in the presence of vitamin D3 (VD3). In summary, human myeloid DC subset differentiation is inversely regulated by GATA-1 and VDR. GATA-1 mediates the repression of VDR and enables IL-4-dependent moDC differentiation. Conversely, VDR is induced downstream of transforming growth factor beta1 and is functionally involved in promoting LC differentiation.

Down-regulation of RXRalpha expression is essential for neutrophil development from granulocyte/monocyte progenitors.

Neutrophil granulocytes (Gs) represent highly abundant and short-lived leukocytes that are constantly regenerated from a small pool of myeloid committed progenitors. Nuclear receptor (NR) family members are ligand-activated transcription factors that play key roles in cellular proliferation and differentiation processes including myelopoiesis. Retinoid X receptor alpha (RXRalpha) represents the predominant NR types I and II homo- and heterodimerization partner in myeloid cells. Here we show that human myeloid progenitors express RXRalpha protein at sustained high levels during macrophage colony-stimulating factor (M-CSF)-induced monopoiesis. In sharp contrast, RXRalpha is down-regulated during G-CSF-dependent late-stage neutrophil differentiation from myeloid progenitors. Down-regulation of RXRalpha is critically required for neutrophil development since ectopic RXRalpha inhibited granulopoiesis by impairing proliferation and differentiation. Moreover, ectopic RXRalpha was sufficient to redirect G-CSF-dependent granulocyte differentiation to the monocyte lineage and to promote M-CSF-induced monopoiesis. Functional genetic interference with RXRalpha signaling in hematopoietic progenitor/stem cells using a dominant-negative RXRalpha promoted the generation of late-stage granulocytes in human cultures in vitro and in reconstituted mice in vivo. Therefore, our data suggest that RXRalpha down-regulation is a critical requirement for the generation of neutrophil granulocytes.

Human Langerhans-cell activation triggered in vitro by conditionally expressed MKK6 is counterregulated by the downstream effector RelB.

Environmentally exposed epithelial Langerhans cells (LCs) encounter diverse innate stress signals, which lead to the activation of complex intracellular signaling cascades. Among these, p38 MAPK is consistently phosphorylated. For which aspects of LC activation triggering of p38 signaling is sufficient remains to be elucidated. We show that conditional induction of a dominant active form of MAPK kinase 6 (d.a.MKK6), a direct upstream kinase of p38, in LCs efficiently induces the up-regulation of costimulatory molecules and enhances their T-cell stimulatory capacity. These immediate effects showed no or only a minor requirement for classical NF-kappaB signaling. Concomitant with LC activation, d.a.MKK6 induced the alternative NF-kappaB member RelB, whose nuclear localization marks mature DCs. Specific inhibition of nuclear RelB during d.a.MKK6-induced LC activation further enhanced their maturation state. This observation was validated using the p38 activator anisomycin, thus suggesting a novel LC intrinsic control mechanism regulated by RelB.

Differential involvement of PU.1 and Id2 downstream of TGF-beta1 during Langerhans-cell commitment.

Langerhans cells (LCs) are highly abundant dendritic cells (DCs) in epidermal and mucosal tissues. The transcription factors PU.1 and Id2 have been implicated as positive regulators of LC development from hematopoietic progenitor cells. LC differentiation from progenitors is absolutely dependent on transforming growth factor beta 1 (TGF-beta1) in vitro as well as in vivo; however, downstream mechanisms are poorly defined. We found that both PU.1 and Id2 are induced by TGF-beta1 in human CD34+ monocyte/LC (M/LC) progenitor cells, and that neither ectopic PU.1 or Id2 alone, nor both together, could replace TGF-beta1 in its instructive function on LC commitment. However, both factors critically contributed to LC differentiation by acting at 2 distinct intersection points. Ectopic PU.1 strongly enhanced TGF-beta1-dependent LC development. Additionally, Notch-induced generation of interstitial-type DCs was associated with PU.1 up-regulation. Thus, PU.1 is generally increased during myeloid DC development. Ectopic Id2 inhibits the acquisition of early monocytic characteristics by cells generated in the absence of TGF-beta1 and also inhibits monocyte induction by alternative stimuli. Since TGF-beta1 represses a default monocyte pathway of common progenitor cells, PU.1 and Id2 seem to modulate lineage options of M/LC precursors, downstream of TGF-beta1.

RelB regulates human dendritic cell subset development by promoting monocyte intermediates.

In humans, epithelial Langerhans cells (LCs) and monocyte-derived/interstitial dendritic cells (DCs) constitute 2 myeloid DC sublineages. Molecular mechanisms involved in their development from common myeloid progenitors remain poorly defined. Here we demonstrate that the nuclear factor-kappaB (NF-kappaB) transcription factor RelB regulates the generation of monocytic CD14(+)CD11b(+) precursors of interstitial DCs from human hematopoietic progenitors. RelB overexpression promoted, whereas endogenous RelB inhibition (by p100DeltaN) blocked, precursor cell development along this DC subset pathway. RelB inhibition specifically arrested precursor progression from CD14(lo)CD11b(-) to CD14(+)CD11b(+) stages. Precursors were still capable of LC and granulocyte differentiation but were defective in macrophage-colony-stimulating factor (M-CSF)-dependent monocyte/macrophage differentiation. RelB inhibition markedly differed from classical NF-kappaB signaling inhibition because IkappaBalpha superrepressor (IkappaBalpha-SR), but not p100DeltaN, impaired LC/DC differentiation, DC adhesion, and progenitor cell proliferation. Although RelB up-regulation and nuclear translocation are regarded as hallmarks of human myeloid DC maturation, ectopic RelB overexpression failed to promote DC maturation. Our results suggest that RelB regulates human monopoiesis and monocyte-derived DC subset development.

Selection of peptide entry motifs by bacterial surface display.

Surface display technologies have been established previously to select peptides and polypeptides that interact with purified immobilized ligands. In the present study, we designed and implemented a surface display-based technique to identify novel peptide motifs that mediate entry into eukaryotic cells. An Escherichia coli library expressing surface-displayed peptides was combined with eukaryotic cells and the gentamicin protection assay was performed to select recombinant E. coli, which were internalized into eukaryotic cells by virtue of the displayed peptides. To establish the proof of principle of this approach, the fibronectin-binding motifs of the fibronectin-binding protein A of Staphylococcus aureus were inserted into the E. coli FhuA protein. Surface expression of the fusion proteins was demonstrated by functional assays and by FACS analysis. The fibronectin-binding motifs were shown to mediate entry of the bacteria into non-phagocytic eukaryotic cells and brought about the preferential selection of these bacteria over E. coli expressing parental FhuA, with an enrichment of 100000-fold. Four entry sequences were selected and identified using an S. aureus library of peptides displayed in the FhuA protein on the surface of E. coli. These sequences included novel entry motifs as well as integrin-binding Arg-Gly-Asp (RGD) motifs and promoted a high degree of bacterial entry. Bacterial surface display is thus a powerful tool to effectively select and identify entry peptide motifs.