Brain transcriptomics reveal the activation of neuroinflammation pathways during acute Orientia tsutsugamushi infection in C57BL/6 mice
We performed RNAseq on total RNA extracted from brains of mice infected with Orientia tsutsugamushi to investigate the transcriptomic signature of this tissue throughout infection. Overall design: Female C57BL/6J mice (6-9 weeks-old, purchased from the Jackson Laboratory #000664) were intravenously inoculated with a lethal dose (6 × 10e4 FFU, 200 µl) of bacteria or PBS (mock). Mice were sacrificed at established timepoints, and spleens were collected in RPMI 1640 medium. Spleen single-cell suspensions were made by passing splenic tissue through a 70-µm cell strainer in RPMI 1640 medium, and these were then treated with Red Blood Cell Lysis Buffer. Untouched B cells were isolated from spleen single-cell suspensions by using the mouse B Cell Isolation Kit (Miltenyi Biotec). B cell samples were then evaluated for purity (~97-98%, based on B220 expression assessed by flow cytometry) and stored in RNALater. Total RNA was extracted by using the RNeasy Mini kit (Qiagen). Total RNA (= 1ug) was sent to LC Sciences (Houston, TX) for Poly(A) RNA-Seq Sequencing Service.The Mus musculus genome build mm10 was used as a reference genome. Quality control analysis was done by using FastQC (Andrews, 2010) and RSeQC6 (Wang, Bioinformatics, 2012). Semi-supervised hierarchical clustering analysis and heatmap generation were performed by using the Morpheus tool. Principal component analysis and volcano plot were done in R using PCAtools and EnhancedVolcano packages.         GSEA analysis    Pathway enrichment analysis using gene set enrichment analysis with GSEA version 3.0 was performed with a ranked gene list (33, 34). Specifically, ranked gene list is prepared from the differential gene expression analysis by using the following metrics: sign of the Log2fold change multiplied by the inverse of the p-value. GSEAPreranked was run with 2000 permutations. Primary gene sets investigated were obtained from the MSigDB and published reports. GSEA FDR Q <0.05 cutoff was applied to examine enriched gene sets in our dataset.         Cytoscape analysis    Enrichment results from the GSEA analysis were visualized using Cytoscape. GSEA output files were uploaded in the EnrichmentMap app of Cytoscape and FDR Q value cut off was set to 0.01. Clusters are automatically defined using AutoAnnotate Cytoscape app.         Statistical Analysis    Data were presented as mean ± standard error of mean (SEM). Differences between individual treatment and control groups were determined by using unpaired Student's t test, utilizing Welch's correction when appropriate. One-way ANOVA was used for multiple group comparisons, with a Tukey's Post Hoc for comparisons between groups. All data were analyzed by using GraphPad Prism software 8.0. Statistically significant values are denoted as *, p < 0.05; **; p < 0.01, ***; p < 0.001, and ****; p < 0.0001, respectively.