Evaluation of organic dust components cytotoxicity on thp1 monocytes-derived-macrophages using high content analysis

Background: Organic dust contains pathogen associated molecular patterns (PAMPs) which can induce, following chronic exposure, significant airway diseases. Mononuclear phagocytes are key protecting cells of the respiratory tract. Several studies have investigated the effects of PAMPs, and mainly endotoxins, on cytokine production. However the sub-lethal cytotoxicity of organic dust components on macrophages has not been tested yet. The novel technology of high content analysis (HCA) is already used to assess subclinical drug-induced toxicity. It combines the capabilities of flow cytometry, intracellular fluorescence probes, and image analysis and enables to perform rapid multiple analysis in large numbers of samples. Objectives: The purpose of the present study was, by using HCA, to investigate the cytotoxicity of the 3 major PAMPs contained in organic dust, ie. endotoxin (LPS), peptidoglycan (PGN) and β-glucans (zymosan) on THP-1 monocyte-derived macrophages. Methods: LPS was used at concentrations of 0.005, 0.01, 0.02, 0.05, 0.1 and 1 μg/ml; PGN and zymosan were used at concentrations of 1, 5, 10, 50, 100 and 500 μg/ml. Cells were exposed to PAMPs for 24 hours. In addition, the oxidative burst and the phagocytic capabilities of the cells were tested. Results: An overlap between PGN intrinsic fluorescence and red/far-red fluorescent dyes occurred, rendering the evaluation of some parameters impossible for PGN. LPS induced sub-lethal cytotoxicity at the lowest dose (from 10 ng/ml). However, the most spectacular changes occurred with zymosan. In addition, zymosan, but not LPS, induced phagosome maturation and oxidative burst. Conclusions: Given the fact that β-glucans can be up to 100 fold more concentrated in organic dust than LPS, these results suggest that β-glucans could play a major role in macrophages impairment following heavy dust exposure and will deserve further investigation in the near future.