Nabil El Zein

MicroRNA Profile of Circulating CD4-positive Regulatory T Cells in Human Adults and Impact of Differentially Expressed MicroRNAs on Expression of Two Genes Essential to Their Function

Background: Regulatory T cells are a subset of T cells with immunosuppressive properties, crucial for immune tolerance, which are also associated with cancer development. Results: The human circulating CD4+ Treg microRNA signature was identified. Conclusion: Differentially expressed microRNAs from the Treg miR signature directly and indirectly regulate crucial Treg genes (FOXP3 and CTLA-4). Significance: Identifying novel regulatory mechanisms of crucial Treg genes expression provides better insight into their biology and offers potential new targets for immunomodulatory therapies. Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4+ Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3′-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3′-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4+ adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3′-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4+ Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.

VIP differentially activates β2 integrins, CR1, and matrix metalloproteinase-9 in human monocytes through cAMP/PKA, EPAC, and PI-3K signaling pathways via VIP receptor type 1 and FPRL1

The neuropeptide vasoactive intestinal peptide (VIP) regulates the exocytosis of secretory granules in a wide variety of cells of neuronal and non‐neuronal origin. In human monocytes, we show that the proinflammatory effects of VIP are associated with stimulation of exocytosis of secretory vesicles as well as tertiary (gelatinase) granules with, respectively, up‐regulation of the membrane expression of the β2 integrin CD11b, the complement receptor 1 (CD35), and the matrix metalloproteinase‐9 (MMP‐9). Using the low‐affinity formyl peptide receptor‐like 1 (FPRL1) antagonist Trp‐Arg‐Trp‐Trp‐Trp‐Trp (WRW4) and the exchange protein directly activated by cAMP (EPAC)‐specific compound 8CPT‐2Me‐cAMP and measuring the expression of Rap1 GTPase‐activating protein as an indicator of EPAC activation, we found that the proinflammatory effect of VIP is mediated via the specific G protein‐coupled receptor VIP/pituitary adenylate cyclase‐activating protein (VPAC1) receptor as well as via FPRL1: VIP/VPAC1 interaction is associated with a cAMP increase and activation of a cAMP/p38 MAPK pathway, which regulates MMP‐9, CD35, and CD11b exocytosis, and a cAMP/EPAC/PI‐3K/ERK pathway, which regulates CD11b expression; VIP/FPRL1 interaction results in cAMP‐independent PI‐3K/ERK activation with downstream integrin up‐regulation. In FPRL1‐transfected Chinese hamster ovary‐K1 cells lacking VPAC1, VIP exposure also resulted in PI‐3K/ERK activation. Thus, the proinflammatory effects of VIP lie behind different receptor interactions and multiple signaling pathways, including cAMP/protein kinase A, cAMP/EPAC‐dependent pathways, as well as a cAMP‐independent pathway, which differentially regulates p38 and ERK MAPK and exocytosis of secretory vesicles and granules.

Valproate Treatment of Human Cord Blood CD4-positive Effector T Cells Confers on Them the Molecular Profile (MicroRNA Signature and FOXP3 Expression) of Natural Regulatory CD4-positive Cells through Inhibition of Histone Deacetylase

Regulatory T cells (Tregs) play a key role in immune system homeostasis and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. Although not sufficient, a high expression of forkhead box P3 (FOXP3) is necessary for their suppressive function. Recent reports have shown that histones deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Tregs CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Tregs signature. Valproate treatment induced binding of Ets-1 and Ets-2 to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs Tregs signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miRs expression profile is not due to an increased expression of FOXP3 but directly results from histones deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in expression level of miR-21 and miR-31. We conclude that valproate treatment of human non-Tregs confers on them a molecular profile similar to that of their regulatory counterpart.

Dendritic Cells Generated in Clinical Grade Bags Strongly Differ in Immune Functionality When Compared With Classical Dcs Generated in Plates

Mature dendritic cells (DCs) represent, by far, the most potent antigen-presenting cells. The development of clinical grade techniques to produce them in large numbers has rendered possible their use in clinical trials. It is therefore crucial to assess the DCs characteristics according to the methodology used to generate them, to improve the comparison and standardization of these trials. We thus compared DCs generated and matured in culture plates (pla-DCs) or in clinical grade bags (bag-DCs) by analyzing, their secretion of bioactive interleukin (IL)-12 and their capacity to induce in-vitro primary responses. We also used several molecular techniques to better characterize the functional differences between the 2 type of DCs. Mature bag-DCs displayed a mature phenotype, but did not secrete significant amounts of IL-12 and failed to initiate primary immune responses. Molecular analyses performed on immature bag-DCs showed them already engaged in a particular maturation process (early activation of nuclear factor κ B and β-catenin). Using microarrays, we found underexpression of receptors for the maturation cocktail in bag-DCs. In mature bag-DCs, we found crucial genes (IL-12, chemokines, and costimulatory and adhesion molecules) down-regulated. Electrophoertic mobility shift assay and Western blots showed a normal activation profile in mature pla-DCs, but not in bag-DCs where the Mek/Erk pathway was still activated. Our results strongly suggest that differentiation of monocytes into DCs in bags generates immature DCs already engaged in an inefficient type of activation, with down-regulation of genes involved in response to the maturation cocktail. This results in mature DCs unable to induce TH1-type responses.

Thyroxine (T4) Transfer from Blood to Cerebrospinal Fluid in Sheep Isolated Perfused Choroid Plexus: Role of Multidrug Resistance-Associated Proteins and Organic Anion Transporting Polypeptides

Thyroxine (T4) enters the brain either directly across the blood–brain barrier (BBB) or indirectly via the choroid plexus (CP), which forms the blood–cerebrospinal fluid barrier (B-CSF-B). In this study, using isolated perfused CP of the sheep by single-circulation paired tracer and steady-state techniques, T4 transport mechanisms from blood into lateral ventricle CP has been characterized as the first step in the transfer across the B-CSF-B. After removal of sheep brain, the CPs were perfused with 125I-T4 and 14C-mannitol. Unlabeled T4 was applied during single tracer technique to assess the mode of maximum uptake (Umax) and the net uptake (Unet) on the blood side of the CP. On the other hand, in order to characterize T4 protein transporters, steady-state extraction of 125I-T4 was measured in presence of different inhibitors such as probenecid, verapamil, BCH, or indomethacin. Increasing the concentration of unlabeled-T4 resulted in a significant reduction in Umax%, which was reflected by a complete inhibition of T4 uptake into CP. In fact, the obtained Unet% decreased as the concentration of unlabeled-T4 increased. The addition of probenecid caused a significant inhibition of T4 transport, in comparison to control, reflecting the presence of a carrier mediated process at the basolateral side of the CP and the involvement of multidrug resistance-associated proteins (MRPs: MRP1 and MRP4) and organic anion transporting polypeptides (Oatp1, Oatp2, and Oatp14). Moreover, verapamil, the P-glycoprotein (P-gp) substrate, resulted in ~34% decrease in the net extraction of T4, indicating that MDR1 contributes to T4 entry into CSF. Finally, inhibition in the net extraction of T4 caused by BCH or indomethacin suggests, respectively, a role for amino acid “L” system and MRP1/Oatp1 in mediating T4 transfer. The presence of a carrier-mediated transport mechanism for cellular uptake on the basolateral membrane of the CP, mainly P-gp and Oatp2, would account for the efficient T4 transport from blood to CSF. The current study highlights a carrier-mediated transport mechanism for T4 movement from blood to brain at the basolateral side of B-CSF-B/CP, as an alternative route to BBB.

fMLP-dependent activation of Akt and ERK1/2 through ROS/Rho A pathways is mediated through restricted activation of the FPRL1 (FPR2) receptor

Objective and designThe objective of this study is to uncover the signal transduction pathways of N-formyl methionyl-leucyl-phenylalanine (fMLP) in monocyte.Materials or subjectsFreshly isolated human peripheral blood monocytes (PBMC) were used for in vitro assessment of signal transduction pathways activated by fMLP.TreatmentTime-course and dose–response experiments were used to evaluate the effect of fMLP along with the specific inhibitors/stimulators on the activation of downstream signaling kinases.MethodsFreshly isolated human PBMC were stimulated with fMLP for the desired time. Western blot and siRNA analysis were used to evaluate the activated intracellular signaling kinases, and flow analysis was performed to assess the levels of CD11b. Furthermore, luminescence spectrometry was performed to measure the levels of released hydrogen peroxide in the media.ResultsfMLP strongly stimulated the activation of AKT and ERK1/2 through a RhoA-GTPase-dependent manner and also induced H2O2 release by monocytes. Furthermore, fMLP mediated its effects through restricted activation of formylpeptide receptor-like 1 (FPRL1/FPR2), but independently of either EGFR transactivation or intracellular calcium release. In addition, NAC reversed fMLP- and H2O2-induced activation of Akt and RhoA-GTPase.ConclusionCollectively, these data suggested that fMLP-activated ERK1/2 and Akt pathways through specific activation of the FPRL1/ROS/RoA-GTPase pathway.

Characterization of a RNA control element that binds p-TEFb and modulates transcription of the human CD3y gene

Our studies show that TCR/CD3 surface receptors are downmodulated after HIV-1, HIV-2 and HTLV-1 infection of CD4+ T cells due to a specific defect in CD3y gene transcripts. Studies of CD3 y transcriptional control revealed parallels with elements regulating HIV-1 gene expression, including a stem loop structure similar to HIV TAR. Analysis of various mutants and deletions in this region revealed that a 43 bp sequence starting from the major transcription start site is critical for positive gene expression. Deletion of ten nucleotides in this region results in a 70% decrease in promoter activity, while deletion of 39 nucleotides completely eliminates promoter activity. EMSA experiments using DNA or RNA probes covering the +1 to +53 region demonstrate that this element functions through an RNA rather than a DNA intermediate. EMSA and Western blots were used to show that this RNA sequence specifically binds the cellular proteins Cyclin-T1 and CDK9 (p-TEFb) as well as the viral transactivator Tat. Deletion of the U at position +9 and U at +37 completely abrogates binding and promoter activity. The p-TEFb-Tat complex is known to promote transcription from the viral LTR, whereas its binding to the CD3y RNA stem loop structure is associated with negative transcriptional regulation. Experiments are currently underway to elucidate mechanisms that regulate the function of these RNA-protein complexes.