Graduation Date

Fall 12-14-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Medical Sciences Interdepartmental Area

First Advisor

Howard S. Fox

Abstract

Toll-like receptors (TLRs) play an important role in the innate immune system. Emerging evidence shows that TLRs, especially endosomal TLRs, can participate in CNS diseases by increasing the production of proinflammatory cytokines via recognition of microRNAs (miRNAs), however which of the miRNAs are able to activate signaling and whether specific sequence motifs are involved remains incompletely defined. Here we found that numerous miRNAs induced TNF-a production across multiple myeloid cell types, including microglia, and that this effect was abolished in cells deficient of TLR7. In particular, miR-20a-5p and miR-148b-3p preferentially stimulate cytokine secretion compared to miR-20b-5p and miR-148a-3p, respectively, despite sharing similar sequences. Further examination of closely related miRNAs that differed in their ability to activate TLR7 resulting in the identification of a motif (UGCUUAU) as well as specific nucleotides (all of the uridines but surprisingly including the cytosine as well), that were vital for secretion of cytokines through TLR7 stimulation. A minimal 10-nucleotide sequence including this motif was found to be the shortest ssRNA that was identified to signal through TLR7. A miRNA containing this motif induced multiple pro-inflammatory molecules, which required the PI3K, MAPK and NF-kB signaling pathways. Wild-type mice that were administered miR-20a-5p (containing this motif) demonstrated increased leukocyte migration. This effect was significantly ameliorated in TLR7 knockout mice, and mice injected with miR-20b-5p (in which the motif is mutated) failed to exert this effect. Our results provide a detailed analysis of miRNAs that activate endosomal TLR7, and identify key nucleotide features of sequence motif recognized by TLR7.

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