MicroRNA and mRNA Transcriptome Profiling in Primary Human Astrocytes Infected with <i>Borrelia burgdorferi</i>

<div><p>Lyme disease is caused by infection with the bacterium <i>Borrelia burgdorferi (Bb)</i>, which is transmitted to humans by deer ticks. The infection manifests usually as a rash and minor systemic symptoms; however, the bacteria can spread to other tissues, causing joint pain, carditis, and neurological symptoms. Lyme neuroborreliosis presents itself in several ways, such as Bell’s palsy, meningitis, and encephalitis. The molecular basis for neuroborreliosis is poorly understood. Analysis of the changes in the expression levels of messenger RNAs and non-coding RNAs, including microRNAs, following <i>Bb</i> infection could therefore provide vital information on the pathogenesis and clinical symptoms of neuroborreliosis. To this end, we used cultured primary human astrocytes, key responders to CNS infection and important components of the blood-brain barrier, as a model system to study RNA and microRNA changes in the CNS caused by <i>Bb</i>. Using whole transcriptome RNA-seq, we found significant changes in 38 microRNAs and 275 mRNAs at 24 and 48 hours following <i>Bb</i> infection. Several of the RNA changes affect pathways involved in immune response, development, chromatin assembly (including histones) and cell adhesion. Further, several of the microRNA predicted target mRNAs were also differentially regulated. Overall, our results indicate that exposure to <i>Bb</i> causes significant changes to the transcriptome and microRNA profile of astrocytes, which has implications in the pathogenesis, and hence potential treatment strategies to combat this disease.</p></div

Selected long non-coding RNAs altered in response to <i>Bb</i>.

<p>Selected long non-coding RNAs altered in response to <i>Bb</i>.</p

Differential expression of microRNAs following exposure to <i>Bb</i>.

<p>MicroRNAs were isolated from astrocytes treated with <i>Bb</i> for 24h (N = 3) and 48h (N = 3), using the microRNA-only procedure at the same time as RNAs, as described in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170961#pone.0170961.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170961#pone.0170961.g003" target="_blank">3</a>. Heat map analysis (A) showing changes in a subset of microRNAs (B) following 48h of <i>Bb</i> treatment. (C) Validation of microRNA changes were performed on 4 microRNAs (see text for details), and revealed upregulation of miR143, miR146b-1, miR199a1 and miR376a2. T-test (* = p-value<0.05; *** = p-value <0.0001).</p

The microRNA-only isolation method for sequencing resulted in a higher percentage of unique reads that mapped to microRNAs.

<p>Astrocytes treated with <i>Bb</i> were lysed, and either total RNA and microRNA, or the microRNA-only fractions were isolated using procedures according to the miRNeasy kit (A). The microRNA from both preparations and libraries made using the Illumina TruSeq small RNA library kit. (B). Libraries were size-selected using the Illumina custom RNA ladder for size selection of the ~145-160bp band, and sequenced using the MiSeq. Lane 1: small RNA marker; lanes 2–3: duplicates of total RNA+miRNA preps; lanes 4–5: duplicates of miRNA only preps (C). The total number of reads, the percent alignments and number of unique reads were comparable in both conditions, but the microRNA-only fraction had a significantly higher percentage of unique reads that mapped to microRNAs relative to the total RNA+microRNA fraction (48% vs 18%). See text for details.</p

MicroRNA differential expression pathway analysis.

<p>We identified pathways targeted by differentially expressed miRNAs using the microT-CDS algorithm in DIANA-miRPath. Heat map of microRNAs vs Pathways, where microRNAs and pathways are clustered using Euclidean distances and complete linkage of binary values (0 = non-significant p-value and 1 = significant p-value). Red squares signify a significant p-value and light yellow signify a non-significant p-value.</p

Selected inflammation and immune function genes altered in response to <i>Bb</i>.

<p>Selected inflammation and immune function genes altered in response to <i>Bb</i>.</p

Pairwise comparison of microRNA and RNA changes.

<p>Pairwise comparison of microRNA and RNA changes.</p