Post-transcriptional down-regulation of Toll-like receptor signaling pathway in umbilical cord blood plasmacytoid dendritic cells
Highlights
► We reveal the post-transcriptional control of IFN-alpha production in cord blood. ► Cord blood plasmacytoid dendritic cells exhibit a profound defect in IFN-a production. ► This defect correlates with the lower expression of proteins within the TLR9 pathway. ► We also observed a higher expression of miR-146a and miR-155. ► Although, cord blood PDC are mature and differentiate in antigen-presenting cells.
Introduction
Allogeneic hematopoietic stem cell transplantation (HSCT) is widely used for treating hereditary and/or hematological conditions of both malignant and non-malignant origin. Umbilical cord blood (UCB) has been used as an alternative source of HSC in transplantation for patients without an HLA-matched related donor, with similar therapeutic success as compared with unrelated bone marrow transplantation [1], [2], [3], [4], [5]. Unlike transplantation with other sources of HSC, UCB transplantation is characterized by a low incidence and severity of graft-versus-host-disease (GvHD), caused by donor T-cells recognizing and reacting to host major and minor histocompatibility antigens [6]. Nonetheless, this low rate of acute GvHD is not associated with a higher rate of leukemia relapse, indicating that graft-versus-leukemia (GvL) mechanisms are effective despite the lower alloreactivity [1], [7]. This contrasts with HSCT using other stem cell sources where increasing the GvL effect is associated with increased GvHD, and it indicates that UCB cells have unique immune properties that allow for allogeneic tolerance while preserving anti-tumoral activity.
Dendritic cells (DCs) capture, process and present antigens to T lymphocytes to induce immunity or tolerance [8]. Both host and donor DC participate in the initiation and the maintenance of GvHD following HSCT: host DC by presenting host-derived antigens to donor-T cells [6], [9], and graft-derived DC by maintaining and expanding alloreactive CD8+ T cells [9], [10], [11]. Several subtypes of DC, namely myeloid DC (mDC), plasmacytoid DC (PDC) and CD14−/lowCD16+ dendritic cells are present in UCB [12], [13]. Functional differences have been described between adult and neonatal DC subsets that may account for some of the unique immune properties of UCB cells [12], [14], [15], [16]. PDC are of particular interest because of their ability to induce either Th1 or Th2-dependent T cell cytotoxic responses and to induce tolerance through regulatory T cell (Treg) differentiation, depending on their environment [17]. It was even shown in a mouse model of hematopoietic transplantation that PDC can enhance the GvL activity of donor T cells without increasing GvHD, reminiscent of what is observed following UCB transplantation [18]. Further characterization of the immune properties of UCB PDC may therefore shed light on the unique immune properties of UCB, and open new avenues for improving UCB transplantation outcome.
PDC selectively express Toll-like receptors 7 and 9 (TLR-7 and TLR-9), which are localized in the endosomal–lysosomal compartment and recognize unmethylated CpG motifs on viral RNA and DNA [19]. Upon activation by TLR-ligands, adult PDC rapidly secrete massive amounts of type I interferons (IFN) as well as chemokines that allow for the recruitment and the activation of immune cells. This rapid and massive IFN response is due to the constitutively high level of expression of the IRF-7 transcription factor, which is phosphorylated and translocated to the nucleus upon activation of the TLR-7 or TLR-9 signaling pathways. PDC then undergo maturation to fully differentiated antigen-presenting cells and up-regulate the expression of T cell co-stimulatory molecules [20]. Type I IFN production is highly regulated by a set of positive and negative feedback loops as well as by cell surface receptors coupled with immunoreceptor tyrosine-based activation motifs (ITAMs) [21]. In addition, the post-transcriptional regulation of TLR-signaling pathway molecules by microRNA miR-146a [22], [23], [24], [25] and miR-155 [26] has been described, including molecules implicated in the control of type I IFN production [22], [27], [28]. MicroRNAs are small non-coding RNAs that suppress gene expression by binding to partially complementary sequences of mRNAs and inhibiting their translation into protein or accelerating their degradation [29].
CB PDC produce much less type I IFN upon activation by viral antigens or TLR-ligands [30], [31], [32], resembling tolerogenic PDC that were described in some tumor environments [33], [34], [35]. Here we examined the immune properties of UCB PDC at the cellular and molecular levels and compared them with adult PDC. Our results reveal that the lower IFNα production by UCB PDC is not due to immaturity but rather could be the result of a down regulation of TLR signaling molecules at the post-transcriptional level. Accordingly, we observed increased levels of miR-146a and miR-155 in UCB PDC as compared with adult PDC.
Section snippets
Blood and bone marrow samples
Cord blood samples were provided by the Research Cord Blood Bank of the CHU Sainte-Justine, in partnership with the public Cord Blood Bank administered by Héma-Québec. Cord blood units were harvested in sodium citrate collection packs by umbilical vein puncture after normal delivery of full-term healthy neonates. Adult peripheral blood samples were collected in heparinized tubes from healthy volunteers. Femoral heads from patients who undergone a total hip replacement surgery were used as a
Differentiation phenotype of UCB PDC
Three stages of differentiation can be distinguished by cell surface marker expression within the HLA-DR+ CD123hi PDC population in human bone marrow. The most immature stage of differentiation (stage I) is characterized by CD34 expression and low CD45 expression. Stage II cells express lower CD34 levels and higher CD45, and stage III cells are CD34 negative CD45hi [38]. We compared these differentiation stages in adult blood and UCB in order to determine the differentiation stage of UCB PDC.
Discussion
The present report explored UCB PDC phenotype and function to get new insights into the mechanisms of higher tolerance to alloantigens of UCB cells. UCB PDC exhibit a fully differentiated surface phenotype, uptake antigens and up-regulate co-stimulatory molecules upon activation, although with delayed kinetics. Their lower IFN-α response is associated with a post-transcriptional down-regulation of TLR-9 signaling pathway proteins. This down-regulation is in line with an increased expression of
Acknowledgments
This work was supported by the Fonds de Recherche en Santé du Québec and the Fondation Charles-Bruneau. Emily Charrier is a fellow of The Cole Foundation and the Fondation du CHU Sainte-Justine/Fondation des Etoiles. We thank Hugo Morin and Dr. Isabelle Louis at the Research Cord Blood Bank of CHU Sainte-Justine for providing cord blood units; Danièle Gagné (Institut de Recherche en Immunologie et Cancer, Université de Montréal) for cell sorting; Laurent Knafo for technical assistance in
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