Figure 1.

Pre-incubation of THP-1 cells with LPS results in a change in immune phenotype consistent with endotoxin tolerance. THP-1 cells were pre-incubated with LPS (100 ng/ml) for 0–72 h and the ability to respond to a second inflammatory stimulus was then determined. (A) THP-1 cells were pre-incubated with 1, 10, or 100 ng/ml LPS for 24 h before measuring release of TNFα during a further 4-h exposure to a second stimulus of 10 or 100 ng/ml LPS by ELISA. (B)The release of pro-inflammatory TNFα and IL-8 in response to a second exposure to LPS (100 ng/ml) was measured by ELISA (n = 6). (C) Phagocytosis of fluorescein-labeled Escherichia coli (E.coli) in 1 h was measured by flow cytometry (n = 4). (D) Cell viability was determined by measuring the proportion of cells excluding propidium iodide following incubation with LPS (100 ng/ml) for 0–72 h (n = 4). (E) THP-1 cells were incubated with LPS (100 ng/ml) for 0–72 h before measuring macrophage differentiation in comparison to a positive control of cells treated with 10 nM PMA for 72 h. Representative images of THP-1 cell morphology on inversion light microscope at 40x magnifications. (F) THP-1 cell adherence to a 6 well plate was determined by cell counts before and after removal of adherent cells using a cell scraper (n = 3). (G) Flow cytometry was used to determine the relative signal intensity for the expression of the markers of macrophage differentiation CD14, CD36, and CD206 compared to the mean in the medium control (n = 3) (H) Representative histograms indicating the level of cell surface CD14 expression measured by flow cytometry. (I) Extracellular acidification rate with and without olygomycin following exposure to LPS (100 ng/ml) for 0–72 h as determined using the Seahorse XF96^e extracellular flux analyser (n = 5). All data are presented as mean ± standard deviation with the values in panel a expressed as relative cytokine release compared to the mean in the medium control; *p < 0.05, **p < 0.01, ***p < 0.001.