Monocytes/macrophages are key players in all phases of physiological and pathological inflammation. To understanding the regulation of macrophage functional differentiation during inflammation, we designed an in vitro model that recapitulates the different phases of the reaction (recruitment, initiation, development, and resolution), based on human primary blood monocytes exposed to sequential changes in microenvironmental conditions. All reaction phases were profiled by transcriptomic microarray analysis. Distinct clusters of genes were identified that are differentially regulated through the different phases of inflammation. The gene sets defined by GSEA analysis revealed that the inflammatory phase was enriched in inflammatory pathways, while the resolution phase comprised pathways related to metabolism and gene rearrangement. By comparing gene clusters differentially expressed in monocytes vs. M1 and vs. M2 macrophages extracted from an in-house created meta-database, it was shown that cells in the model resemble M1 during the inflammatory phase and M2 during resolution. The validation of inflammatory and transcriptional factors by qPCR and ELISA confirmed the transcriptomic profiles in the different phases of inflammation. The accurate description of the development of the human inflammatory reaction provided by this in vitro kinetic model can help in identifying regulatory mechanisms in physiological conditions and during pathological derangements.

Upregulation of specific transcription factors is a generally accepted mechanism to explain the commitment of hematopoietic stem cells along precise maturation lineages. Based on this premise, transduction of primary hematopoietic stem/ progenitor cells with viral vectors containing the investigated transcription factors appears as a suitable experimental model to identify such regulators. Although MafB transcription factor is believed to play a role in the regulation of monocytic commitment, no demonstration is, to date, available supporting this function in normal human hematopoiesis. To address this issue, we retrovirally transduced cord blood CD34 þ hematopoietic progenitors with a MafB cDNA. Immunophenotypic and morphological analysis of transduced cells demonstrated the induction of a remarkable monomacrophage differentiation. Microarray analysis confirmed these findings and disclosed the upregulation of macrophage-related transcription factors belonging to the AP-1, MAF, PPAR and MiT families. Altogether our data allow to conclude that MafB is a key regulator of human monocytopoiesis.

In spite of their apparently restricted differentiation potentiality, hematopoietic precursors are plastic cells able to transdifferentiate from a maturation lineage to another. To better characterize this differentiation plasticity, we purified CD14À and CD14 þ myeloid precursors generated by 'in vitro' culture of human CD34 þ hematopoietic progenitors. Morphological analysis of the investigated cell populations indicated that, as expected, they consisted of granulocyte and monocyte precursors, respectively. Treatment with differentiation inducers revealed that CD14À cells were bipotent granulo-monocyte precursors, while CD14 þ cells appeared univocally committed to a terminal macrophage maturation. Flow cytometry analysis demonstrated that the conversion of granulocyte precursors to the mono-macrophage maturation lineage occurs through a differentiation transition in which the granulocyte-related myeloperoxidase enzyme and the monocyte-specific CD14 antigen are coexpressed. Expression profiling evidenced that the observed trans-differentiation process was accompanied by a remarkable upregulation of the monocyte-related MafB transcription factor.

2005;23:496-506

No abstract

Background/Aims: Many potentially toxic molecules accumulate in the blood during hepatic dysfunction. In clinical practice, it is very difficult to remove bilirubin, the most widely studied toxin, and particularly the unconjugated form, strongly albumin-bound. The aim of this in vitro study was to assess irreversible bilirubin adsorption as a protein-bound compound marker, using Cytosorb® (Cytosorbents Corp.), a new hemoadsorption device designed to remove cytokines. Methods: We performed 4 in vitro experiments, dynamic and static, with different albumin-bilirubin solutions. Results: All experiments showed the resin’s ability to break the albumin-bilirubin complex (Experiment 1, 2), leading to efficient bilirubin removal for 24 h (Removal Rate: 90% Experiment 3) with minimal albumin loss. No sign of bilirubin release from the charged resin was detected (Experiment 4). Conclusion: Cytosorb® seems a promising artificial liver support, thanks to its ability to adsorb bilirubin and its proven ability to modulate the cytokines involved in hepatic dysfunction.

CD271 is the low-affinity neurotrophin (p75NTR) receptor that belongs to the tumor necrosis factor receptor superfamily. Because in human epidermis, CD271 is predominantly expressed in transit-amplifying (TA) cells, we evaluated the role of this receptor in keratinocyte differentiation and in the transition from keratinocyte stem cells (KSCs) to progeny. Calcium induced an upregulation of CD271 in subconfluent keratinocytes, which was prevented by CD271 small interfering RNA. Furthermore, CD271 overexpression provoked the switch of KSCs to TA cells, whereas silencing CD271 induced TA cells to revert to a KSC phenotype, as shown by the expression of β1-integrin and by the increased clonogenic ability. CD271(+) keratinocytes sorted from freshly isolated TA cells expressed more survivin and keratin 15 (K15) compared with CD271(-) cells and displayed a higher proliferative capacity. Early differentiation markers and K15 were more expressed in the skin equivalent generated from CD271(+) TA than from those derived from CD271(-) TA cells. By contrast, late differentiation markers were more expressed in skin equivalents from CD271(-) than in reconstructs from CD271(+) TA cells. Finally, skin equivalents originated from CD271(-) TA cells displayed a psoriatic phenotype. These results indicate that CD271 is critical for keratinocyte differentiation and regulates the transition from KSCs to TA cells.

ZFP36L1 negatively regulates erythroid differentiation of human hematopoietic progenitors by directly binding the 3′ UTR of Stat5b mRNA, thereby triggering its degradation. This study shows that posttranscriptional regulation is involved in the control of hematopoietic differentiation.