High-Content Screening Technology Combined with a Human Granuloma Model as a New Approach To Evaluate the Activities of Drugs against Mycobacterium tuberculosis

Abstract : Tuberculosis remains a major health problem due to the emergence of drug-resistant strains of Mycobacterium tuberculosis. Some models have provided valuable information about drug resistance and efficacy; however, the translation of these results into effective human treatments has mostly proven unsuccessful. In this study, we adapted high-content screening (HCS) technology to investigate the activities of antitubercular compounds in the context of an in vitro granuloma model. We observed significant shifts in the MIC 50 s between the activities of the compounds under extracellular and granuloma conditions. T uberculosis (TB) is a respiratory disease that is one of the major causes of mortality and morbidity worldwide. Almost 20 people develop TB and four patients die from the disease every minute somewhere in the world (1). The disease can be cured by drug treatment involving a regimen of several drugs. The World Health Organization (WHO) estimates that there were around 0.5 million new cases of multidrug-resistant TB (MDR-TB) in 2012 (2, 3). A recent report confirmed that MDR-TB is a continuing worldwide health problem, even in high-income countries with a low incidence of TB (4). The immune system undoubtedly plays a major role in TB control (5). Signaling events of the immune system lead to the formation of a granuloma, the hallmark of TB. Granulomas are an immune microenvironment in which the infection can be controlled but also a niche in which bacilli can thrive and modulate immune responses to ensure their survival for long periods without causing damage (6, 7). Granulomas are cell aggregates that form tridimensional and heterogeneous structures (8, 9). We previously described an in vitro granuloma model involving human blood cells either infected with mycobacteria or incubated with mycobacterial antigens (8, 10). Either of these models resulted in the formation of a typical epithelioid granuloma with morphological characteristics and properties of cellular differentiation similar to those of natural granulomas (8, 10, 11). This model may help bridge the gap between the in vitro evaluation of MICs and costly in vivo efficacy studies in guinea pigs. In particular, compounds that are effective in the in vitro granuloma model could be prioritized for guinea pig studies. High-content screening (HCS) technology has been used to identify anti-TB compounds (12–14). This technology has several advantages over traditional phenotypic screening approaches. This technology has mostly been used in single-cell experiments, because it is adapted to identify and analyze images (12, 13); however , in this paper, we focus on the use of granulomas instead of single cells. In this report, we describe the development of a novel HCS method to evaluate the activities of reference compounds against a green fluorescent protein (GFP)-expressing H37Rv strain of Mycobacterium tuberculosis (MTB-GFP) within 5 days, and we compared the MIC 50 values with those of classical liquid cultures (extracellular), intracellular growth (single-cell assay), and within granulomas, all using the same bacterial strain. MTB-GFP strains (13) were grown for 3 weeks at 37°C until the log phase was reached. The test inoculum was prepared by diluting the culture with phosphate-buffered saline (PBS) medium or complete RPMI (RPMIc) medium (containing RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum [FBS], with 1% glutamine, 1% HEPES, 1% nonessential amino acids) to a final bacterial concentration of 1 ϫ 10 6 CFU/ml. In vitro human granuloma formation was performed by incu-bating peripheral blood mononuclear cells (PBMC) of healthy volunteers; the cells were purified by gradient density sedimenta-tion (15) with MTB-GFP using an appropriate multiplicity of infection in RPMIc medium at 37°C with low shaking (125 rpm) for 1 h; they were then washed with PBS containing 2% FBS and suspended in RPMIc medium at a concentration of 3.5 ϫ 10 5 cells per well in 384-well plates (Greiner). The cells were then incu-bated for 3 days at 37°C in a 5% CO 2 atmosphere to allow the formation of granulomas. Intragranuloma bacterial quantification was performed by fixing granulomas with 4% paraformaldehyde (Electron Microscopy Sciences) in PBS and staining with Hoechst (Sigma); each step consisted of a 30-min incubation period at room temperature.
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Antimicrobial Agents and Chemotherapy, American Society for Microbiology, 2015, 59 (1), pp.693. 〈10.1128/AAC.03705-14〉
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Mayra Silva-Miranda, Euloge Ekaza, Adrien Breiman, Karim Asehnoune, David Barros-Aguirre, et al.. High-Content Screening Technology Combined with a Human Granuloma Model as a New Approach To Evaluate the Activities of Drugs against Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy, American Society for Microbiology, 2015, 59 (1), pp.693. 〈10.1128/AAC.03705-14〉. 〈inserm-01285011〉

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