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Gene expression profile of differentially recognized Mtb-epitopes as a function of disease history

ABSTRACT: Purpose: The goals of this study are to compare two types of Mtb epitope-specific transcriptome profiling (RNA-seq) in individuals with latent TB infection. Methods: Epitope-specific T cells were isolated by IFNg capture assay or activation induced marker assay (CD25, OX40). Total RNA was purified using miRNAeasy micro kit from Qiagen and amplified using Smart-seq2 protocol. Nextera CT sequencing libraries were prepared and samples were sequenced using HiSeq2500 from Illumina. The sequence reads that passed quality filters were mapped against the hg19 reference using TopHat. Differentially expressed genes between the two epitope sets were identified by Bioconductor package DESeq2, pairwise comparisons and Benjamimi-Hochberg-adjusted p values. Results: Quantile normalized gene expression values and pairwise comparisons between the epitope-specific memory T cells revealed 9 differentially expressed genes (adjusted p<0.05) for IFNg capture and 1 differentially expressed gene for activation induced marker assay.

ORGANISM(S): Homo sapiens

PROVIDER: GSE99668 | GEO | 2017/12/31

SECONDARY ACCESSION(S): PRJNA389273

REPOSITORIES: GEO

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Project description:Microbial keratitis is a major cause of blindness worldwide. An excessive host inflammatory response can occur even after adequate antimicrobial treatment. This results in tissue damage with corneal thinning and even perforation, which may require corneal transplantation. In this study we investigated the pathways involved in the pathophysiology of this disease by comparing the human transcriptome profile of tissue from culture-proven bacterial and fungal keratitis (n=7 and n=8 respectively) with normal non-infected cadaveric corneal tissue (C, n=12) using Illumina HT12 v4 microarrays. The causative organisms were Streptococcus pneumoniae (n=6) and Pseudomonas aeruginosa (n=1) for bacterial keratitis (BK). Fungal keratitis (FK) was caused by Fusarium sp. (n=5), Aspergillus sp. (n=2, A. flavus and terreus) and Lasiodiplodia sp. (n=1). Differential expression (DE) analysis revealed 2310 significantly altered probes in the BK v C comparison, and 1813 probes for FK v C. The most highly upregulated gene in both comparisons was MMP9 with fold changes (FC) of 64 (fdr-adjusted p<6 x10-11) for FK v C and 89 for BK v C (fdr-adjusted p<4 x10-11) respectively. Network co-expression analyses revealed the defense response, inflammatory response and extracellular matrix mechanisms to be the main functional pathways involved. Microarray results were validated by performing real-time quantitative PCR (RTqPCR) for 46 DE genes using RNA extracted from the same samples. There was a high correlation between log2 FC values from microarray and RTqPCR. Further studies are needed to evaluate the most highly differentially expressed genes as possible biomarkers of disease progression or therapeutic targets. Case - control study design. Corneal ulcer tissue from 8 bacterial and 9 fungal ulcers was excised at the time of corneal transplantation surgery and immediately preserved in RNALater. Non-infected corneal tissue from 13 cadaver corneas were the control tissue. Transcriptome profile generated using Illumina HT12 v4 beadchips. Differential expression analysis was performed with pairwise comparisons: bacterial ulcers versus controls, fungal ulcers versus controls and bacterial versus fungal ulcers. Microarray results validated with RTqPCR.

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Gene expression profile of differentially recognized Mtb-epitopes as a function of disease history

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