Identification of microRNAs involved in the development and function of follicular dendritic cells
Aungier, Susan Rebecca
Follicular dendritic cells (FDCs) are key elements of secondary lymphoid organs where they form the stromal component of B-cell follicles. FDCs possess extensive dendritic process that trap intact antigen via Fc and complement receptors on the cell surface. The antigen is displayed to B-cells, providing a basis for selection of high affinity B cells. FDC also have important roles in facilitating the clearance of apoptotic B cells by the secretion of the opsonising factor MFGE8. It is well established that lymphotoxin signalling is required for FDC maturation but the specific details of the molecular mechanisms that regulate FDC development and differentiation are not fully understood. MicroRNAs (miRNAs) are non-coding RNAs of approximately 18-25 nucleotides in length that regulate gene expression at the post-transcriptional level. MiRNAs bind to their target gene transcripts as part of the RNA induced silencing complex and repress translation of the target gene product. The objective of this study was to identify miRNAs that play a role in the development and function of FDCs. An in vivo murine model of FDC de-differentiation was used to provide material for miRNA analysis. By comparison of miRNA profiles from spleen tissue with FDC at different stages of de-differentiation, we would be able to obtain a miRNA signature for mature FDC. Spleens were collected at various time points over a 28 day period following transient blockade of lymphotoxin signalling. A variety of methods were used to profile the miRNAs expressed at different time points during the suppression and recovery of the FDC network. Comparison of the miRNA profiles of spleens containing mature, partially de-differentiated, and fully de-differentiated FDC identified a number of miRNAs that were differentially expressed during FDC de-differentiation. To assess the role of specific miRNAs in FDC development, the mouse FDC-like cell line, FL-YB, was used as an in vitro model system. FL-YB cells were used to perform gain-offunction and loss-of-function studies on selected miRNAs and to assess the effects of various stimuli/conditions on miRNA expression. The effects of different treatments on cell proliferation, morphology and adhesion, and on gene expression by FL-YB, were monitored. Loss-of-function studies for one of the selected miRNA (miR-100-5p) revealed a significant effect on a number of gene transcripts involved in mediation of the germinal centre response (Il-6, Tlr4, Ptgs1/2). These data indicate that miR-100-5p has a role in regulating Il-6, Tlr4 and Ptgs1/2 transcripts. None of these transcripts contain predicted target sites for miR-100-5p and so the effect of miR-100-5p on these transcripts is likely to be indirect. Further studies on these miRNA: target interactions are required to elucidate the mechanisms and biological consequences of miRNA regulation in FDCs.