Abstract
MicroRNA-155 (miR-155) has been involved in the response to inflammation in macrophages and lymphocytes. Here we show how miR-155 participates in the maturation of human dendritic cells (DC) and modulates pathogen binding by down-regulating DC-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN), after directly targeting the transcription factor PU.1. During the maturation of DCs, miR-155 increases up to 130-fold, whereas PU.1 protein levels decrease accordingly. We establish that human PU.1 is a direct target for miR-155 and localize the target sequence for miR-155 in the 3'-untranslated region of PU.1. Also, overexpression of miR-155 in the THP1 monocytic cell line decreases PU.1 protein levels and DC-SIGN at both the mRNA and protein levels. We prove a link between the down-regulation of PU.1 and reduced transcriptional activity of the DC-SIGN promoter, which is likely to be the basis for its reduced mRNA expression, after miR-155 overexpression. Finally, we show that, by reducing DC-SIGN in the cellular membrane, miR-155 is involved in regulating pathogen binding as dendritic cells exhibited the lower binding capacity for fungi and HIV protein gp-120 when the levels of miR-155 were higher. Thus, our results suggest a mechanism by which miR-155 regulates proteins involved in the cellular immune response against pathogens that could have clinical implications in the way pathogens enter the human organism.
Highlights
The role of microRNAs is being intensively studied in many different fields such as fetal development and the immune system
It has been shown previously that most of these inflammatory stimuli up-regulate miR-155 levels in macrophages by activating the NF-B signaling pathway [3, 22]. To determine whether this up-regulation occurs during dendritic cell maturation, we exposed monocyte-derived dendritic cells (DC) to LPS, which is widely reported to drive DC maturation [23]
During LPS-induced maturation, we could detect increased differential expression of miR-155 by 6 h, whereas a maximum value of 136-fold increase was reached by 48 h; no significant change occurred in the immature DCs over that time (Fig. 1A)
Summary
Dendritic cells were generated from human peripheral blood mononuclear cells as previously described [20]. To generate the THP1–155 cell line, HEK293 T cells were transfected with Superfect (Qiagen) following the manufacturer’s protocol with 5 g of pLVTHM_BIC (or pLV/ tTR_KRAB_Red in the case of generating the repressor lentiviral particles), 3.75 g of pPAX2 and 1.5 g of pMD2G. Supernatant from these cells, containing lentiviral particles, was added to THP-1 cells that were preincubated in the presence of 8 g/ml of Polybrene (Sigma) for 30 min at 37 °C. To assess direct targeting of miR-155, pRLTK, pRLTK_WT_3ЈUTR_PU1, or pRLTK_MUT_3ЈUTR_ PU1 were transfected into HeLa cells employing Superfect (Qiagen) following the manufacturer’s instructions.
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