Dataset Information

Expression of inflammatory host genes in Chlamydia trachomatis-infected human monocytes.

ABSTRACT: The aim of this study was to perform a comprehensive gene expression analysis of cytokines, chemokines, and their receptors in Chlamydia trachomatis-infected human monocytes in order to elucidate molecular aspects of their involvement in the host response. Peripheral blood mononuclear cells from three healthy donors were separated and infected with C. trachomatis elementary bodies serovar K (UW/31/Cx) at a multiplicity of infection of 5:1. Three time points of infection were studied by gene expression analysis using microarray: 4 hours (active infection), 1 day (transition), and 7 days (persistent infection). Expression levels of selected genes were confirmed by quantitative real-time reverse transcription-polymerase chain reaction. Transcripts encoding 10 cytokines, chemokines, and receptors were found to be upregulated exclusively in the early, active phase of the infection as compared to four genes in the late, persistent state of the infection. Apart from receptors, both the level and the number of transcripts encoding inflammatory products decreased with ongoing infection. Four genes (interferon-gamma, macrophage inflammatory protein [MIP]-1-alpha, MIP-1-beta, and interleukin-2 receptor-gamma) were constantly expressed over a period of 7 days. The current study provides data on the induction of mRNA encoding cytokines, chemokines, and their receptors in C. trachomatis-infected human monocytes. This pro-inflammatory gene expression profile of the monocytic host cell showed several differences between active and persistent chlamydial infections.

SUBMITTER: Schrader S

PROVIDER: S-EPMC2206345 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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Expression of inflammatory host genes in Chlamydia trachomatis-infected human monocytes.

Schrader Sina S Klos Andreas A Hess Simone S Zeidler Henning H Kuipers Jens G JG Rihl Markus M

Arthritis research & therapy 20070101 3

The aim of this study was to perform a comprehensive gene expression analysis of cytokines, chemokines, and their receptors in Chlamydia trachomatis-infected human monocytes in order to elucidate molecular aspects of their involvement in the host response. Peripheral blood mononuclear cells from three healthy donors were separated and infected with C. trachomatis elementary bodies serovar K (UW/31/Cx) at a multiplicity of infection of 5:1. Three time points of infection were studied by gene expr ...[more]

PMID: 17524146

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Project description:Messenger RNA of C trachomatis infected monocytes of the three healthy donors was compared with the corresponding mock-infected samples. For mock infection, SPG buffer was used instead of the C trachomatis suspension, all other procedures were performed identically. Cells were resuspended in solution D and total RNA was extracted by application of phenol:chloroform 5:1 (pH 4.5, Ambion, Austin, TX) followed by precipitation in isopropanol at -80°C for 1 hr and incubation with RQ1 DNase (Promega, Madison, WI) at 37 °C for 1 hr. The signal intensity of the G3PDH housekeeping gene on the microarray membrane was used as indirect quality marker for the RNA utilized as only experiments were included in the analysis that revealed a G3PDH signal intensity within 1.5 times the standard deviation of all membranes evaluated in our study. The entire microarray procedure and its analysis has recently been validated and reported in detail. Total RNA of all donors was pooled and for each microarray experiment 150 µg were reverse transcribed and amplified by SMART™-PCR (BD Biosciences Clontech, Palo Alto, CA), a technology that allows reverse transcription of small amounts of total RNA and subsequent amplification of the entire cDNA. Probes were labeled with 32P and subsequently over night hybridized to a filter-based nylon membrane containing immobilized cDNA-specific sequences from a total of 1,184 genes (Human Atlas Array 1.2, BD Biosciences Clontech, Palo Alto, CA). For analysis, we focused solely on the 159 cytokines, chemokines and their receptors as given by the manufacturer. Signal intensities were retrieved by a STORM 860 scanner (Molecular Dynamics, Sunnyvale, CA) in combination with the AtlasImage 2.0 software (BD Biosciences Clontech). Data from all signal intensities were then subtracted by the local background intensity measured around each gene. The local background intensities of all individual genes were subsequently averaged, resulting in the mean background intensity of a particular membrane. Gene expression in our experiments was determined by a spot intensity of a single transcript that exceeded twice the mean background. Normalisation to background and to the G3PDH housekeeping gene was achieved by the global (sum) normalization method. Signal intensity data are given as the ratio of C trachomatis infected vs mock infected probes. Keywords: expression analysis

Dataset Information

Expression of inflammatory host genes in Chlamydia trachomatis-infected human monocytes.

Publications

Expression of inflammatory host genes in Chlamydia trachomatis-infected human monocytes.

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