Staining of Chlamydia trachomatis elementary bodies: A suitable method for identifying infected human monocytes by flow cytometry

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Abstract

Persistence of Chlamydia trachomatis (C. trachomatis) in the joint is the most frequent cause of reactive arthritis following urogenital tract infection. The resulting changes of host cell antigen- and cytokine-expression are not precisely understood. We developed and evaluated a direct cytometric approach to visualize in vitro C. trachomatis-infected monocytes. Infectious elementary bodies (EBs) of C. trachomatis serovar K were labelled by incubation with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). Afterwards, human peripheral blood monocytes were cultured with the CFSE-labelled EBs and analysed by flow cytometry. Real-time polymerase chain reaction (PCR) was used to demonstrate intracellular uptake and viability of CFSE-labelled C. trachomatis by the determination of gene expression. Labelling EBs with CFSE may become a valuable tool for studying the interaction between C. trachomatis and the host cell.

Introduction

Chlamydia trachomatis is an important obligate intracellular bacterium that causes trachoma and various urogenital infections. It is one of the most common sexually transmitted pathogens in the world (Beagley and Timms, 2000). During the biphasic developmental cycle, there are two distinct morphological forms: the replicative, intracellular reticulate bodies (RBs) and the extracellular infectious elementary bodies (EBs). During the intracellular phase, RBs multiply by binary fission until the late phase of infection, when they then begin to convert back to EBs. Research from many groups has demonstrated that under some conditions, C. trachomatis gives rise to persistent infection (Beatty et al., 1994). In this state, the organism displays a transcriptional profile that is significantly different from that of actively growing chlamydial cells. Persistence of C. trachomatis in the joint is the most frequent cause of reactive arthritis. During chronic and/or persistent infections, C. trachomatis can be found in low numbers in synovial fibroblasts but are preferentially observed in monocytes and macrophages of patients suffering from reactive arthritis (Beutler et al., 1994, Gerard et al., 2006, Inman et al., 2000, Koehler et al., 1997, Nanagara et al., 1995). Monocytes can spread chlamydiae from the primary site of infection to distant sites. Although monocytes represent the vehicle for chlamydiae, the study of chlamydial infected monocytes has been limited due to the difficulty of specifically analysing monocytes infected in vitro. Rather, heterogeneous populations of infected and non-infected cells have been examined. Therefore, we have developed a technique that allows chlamydial EBs to be labelled with CFSE prior to cell infection and thus allowing identification of chlamydial inclusions in human monocytes by flow cytometry.

Section snippets

Culture and propagation of C. trachomatis

C. trachomatis EBs serovar K (UW/31/Cx; Washington Research Foundation, Seattle, WA, USA) were prepared as previously described (Schmitz et al., 1993). Quantitative real-time PCR was used to determine the absolute number (± standard deviation) of chlamydial organisms in relation to one IFU in a single stock preparation. Contamination with Mycoplasma was excluded by routine assessment using culture and PCR techniques followed by restriction enzyme digestion (Krausse-Opatz et al., 2000).

Generation of human monocytes

Peripheral

CFSE-staining of C. trachomatis and optimization for flow cytometry

EBs were initially stained with several concentrations of CFSE (5, 10, 20 and 50 μmol/l) for different durations (30 min to 2 h). Optimal staining of EBs was observed using 20 μmol/l CFSE for 1.5 h (Fig. 1A). Flow cytometry demonstrated that CFSE-labelled chlamydial EBs were internalized in human monocytes as early as 4 h postinfection (p.i.). However, the signal declined from 27% to 21% after 7 days (Fig. 1B). The intracellular location of the chlamydiae was confirmed by fluorescent microscopy

Discussion

The main goal of the present investigation was to establish a flow cytometric method to specifically detect C. trachomatis-infected monocytes. Previous flow cytometry-based studies of the viability of Escherichia coli and human lymphocyte division have utilized the fluorescent dye CFSE (Lyons and Parish, 1994, Tanaka et al., 2000). One advantage of CFSE is that it is non-toxic to the cells. Furthermore, as labelling can be done without prior fixation, the technique can be performed with viable

Acknowledgements

Supported by a grant from the German Kompetenznetz Rheuma and DFG KU1182/1–3. We are grateful to Bernhard Vaske for his valuable assistance with statistical analysis.

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The first and the second authors contributed equally to this manuscript.

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