Cholera toxin inhibits IL-12 production and CD8α+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function

Prior studies have demonstrated that cholera toxin (CT) and other cAMP-inducing factors inhibit interleukin (IL)-12 production from monocytes and dendritic cells (DCs). We show that CT inhibits Th1 responses in vivo in mice infected with Toxoplasma gondii. This correlated with low serum IL-12 levels and a selective reduction in the numbers of CD8α+ conventional DCs (cDCs) in lymphoid organs. CT inhibited the function of interferon (IFN) regulatory factor (IRF) 8, a transcription factor known to positively regulate IL-12p35 and p40 gene expression, and the differentiation of CD8α+ and plasmacytoid DCs (pDCs). Fluorescence recovery after photobleaching analysis showed that exposure to CT, forskolin, or dibutyryl (db) cAMP blocked LPS and IFN-γ–induced IRF8 binding to chromatin. Moreover, CT and dbcAMP inhibited the binding of IRF8 to the IFN-stimulated response element (ISRE)–like element in the mouse IL-12p40 promoter, likely by blocking the formation of ISRE-binding IRF1–IRF8 heterocomplexes. Furthermore, CT inhibited the differentiation of pDCs from fms-like tyrosine kinase 3 ligand–treated bone marrow cells in vitro. Therefore, because IRF8 is essential for IL-12 production and the differentiation of CD8α+ cDCs and pDCs, these data suggest that CT and other Gs-protein agonists can affect IL-12 production and DC differentiation via a common mechanism involving IRF8

EVI1 Impairs myelopoiesis by deregulation of PU.1 function

EVI1 is an oncogene inappropriately expressed in the bone marrow (BM) of approximately 10% of myelodysplastic syndrome (MDS) patients. This disease is characterized by severe anemia and multilineage myeloid dysplasia that are thought to be a major cause of mortality in MDS patients. We earlier reported on a mouse model that constitutive expression of EVI1 in the BM led to fatal anemia and myeloid dysplasia, as observed in MDS patients, and we subsequently showed that EVI1 interaction with GATA1 blocks proper erythropoiesis. Whereas this interaction could provide the basis for the erythroid defects in EVI1-positive MDS, it does not explain the alteration of myeloid differentiation. Here, we have examined the expression of several genes activated during terminal myelopoiesis in BM cells and identified a group of them that are altered by EVI1. A common feature of these genes is their regulation by the transcription factor PU.1. We report here that EVI1 interacts with PU.1 and represses the PU.1-dependent activation of a myeloid promoter. EVI1 does not seem to inhibit PU.1 binding to DNA, but rather to block its association with the coactivator c-Jun. After mapping the PU.1-EVI1 interaction sites, we show that an EVI1 point mutant, unable to bind PU.1, restores the activation of PU.1-regulated genes and allows a normal differentiation of BM progenitors in vitro

Point mutations in two EVI1 Zn fingers abolish EVI1-GATA1 interaction and allow erythroid differentiation of murine bone marrow cells.

EVI1 is an aggressive nuclear oncoprotein deregulated by recurring chromosomal abnormalities in myelodysplastic syndrome (MDS). The expression of the corresponding gene is a very poor prognostic marker for MDS patients and is associated with severe defects of the erythroid lineage. We have recently shown that the constitutive expression of EVI1 in murine bone marrow results in a fatal disease with features characteristic of MDS, including anemia, dyserythropoiesis, and dysmegakaryopoiesis. These lineages are regulated by the DNA-binding transcription factor GATA1. EVI1 has two zinc finger domains containing seven motifs at the N terminus and three motifs at the C terminus. Supported by results of assays utilizing synthetic DNA promoters, it was earlier proposed that erythroid-lineage repression by EVI1 is based on the ability of this protein to compete with GATA1 for DNA-binding sites, resulting in repression of gene activation by GATA1. Here, however, we show that EVI1 is unable to bind to classic GATA1 sites. To understand the mechanism utilized by EVI1 to repress erythropoiesis, we used a combination of biochemical assays, mutation analyses, and in vitro bone marrow differentiation. The results indicate that EVI1 interacts directly with the GATA1 protein rather than the DNA sequence. We further show that this protein-protein interaction blocks efficient recognition or binding to DNA by GATA1. Point mutations that disrupt the geometry of two zinc fingers of EVI1 abolish the protein-protein interaction, leading to normal erythroid differentiation of normal murine bone marrow in vitro

Conversion of human fibroblasts into monocyte-like progenitor cells

International audienceReprogramming technologies have emerged as a promising approach for future regenerative medicine. Here, we report on the establishment of a novel methodology allowing for the conversion of human fibroblasts into hematopoietic progenitor-like cells with macrophage differentiation potential. SOX2 overexpression in human fibroblasts, a gene found to be upregulated during hematopoietic reconstitution in mice, induced the rapid appearance of CD34+ cells with a concomitant upregulation of mesoderm-related markers. Profiling of cord blood hematopoietic progenitor cell populations identified miR-125b as a factor facilitating commitment of SOX2-generated CD34+ cells to immature hematopoietic-like progenitor cells with grafting potential. Further differentiation toward the monocytic lineage resulted in the appearance of CD14+ cells with functional phagocytic capacity. In vivo transplantation of SOX2/miR-125b-generated CD34+ cells facilitated the maturation of the engrafted cells toward CD45+ cells and ultimately the monocytic/macrophage lineage. Altogether, our results indicate that strategies combining lineage conversion and further lineage specification by in vivo or in vitro approaches could help to circumvent long-standing obstacles for the reprogramming of human cells into hematopoietic cells with clinical potential