Fig 1.
Chronic iron overload increases intracellular granularity and iron content in TDM cells.
(A) Cell viability of THP-1 and TDM cells cultured in media with different concentrations of FeSO4 for 24 and 48 h was assayed by propidium iodide staining and flow cytometry analysis. (B) After iron exposure, I-TDM cells revealed higher side-scatter intensity than TDM cells, while their forward scatter intensities were comparable. Dot blots are presented for cellular side scatter and forward scatter intensities during flow cytometric analysis of THP-1 and TDM cells with or without chronic iron exposure. (C) A comparative study of CD11c and CD86 expression in THP-1 and TDM cells that cultured under normal iron or chronic iron overload conditions. Flow cytometric analysis of the expression of the indicated surface molecules on THP-1, I-THP-1, TDM, and I-TDM cells was performed. Staining profiles (green lines) are shown for the indicated surface molecules. The thin lines indicate isotype controls. (D) Iron uptake (ferrous and/or ferric ion) of THP-1, TDM, I-THP-1, and I-TDM cells were measured using an Iron Assay Kit. The data represent the average of triplicate experiments and the error bars indicate the standard error of the mean. Significance is denoted as follows: * p < 0.05; *** p < 0.001.
Fig 2.
Chronic iron overload impairs the bactericidal activity of macrophages.
(A) The bactericidal activity assay was performed by incubating TDM cells, I-TDM cells, and 50μM chloroquine (CQ)-pretreated TDM cells with E. coli DH5α at 37°C for 0, 2, 4 and 8 h. The bacterial killing rate is presented as the percentage of colony forming units (CFUs) released from cells without incubation (0 h) to the CFUs released from cells sampled at different time points of incubation. (B) The bactericidal activity assay was performed by incubating untreated and DFO-pretreated (100 μM for 12 h) TDM and I-TDM cells with E. coli DH5α at 37°C for 4 h. (C) The bactericidal activity assay was performed by incubating TDM, I-TDM cells, hMDM and I-hMDM with Pseudomonas aeruginosa at 37°C for 4 h. (D) The phagocytic activity of TDM and I-TDM cells was assessed by measuring the phagocytosis of labeled E. coli BioParticles. (E) TDM and I-TDM cells were harvested and incubated with 10 μM DCFDA for 15 min and then analyzed by flow cytometry. The data represent the average of triplicate experiments and the error bars indicate the standard error of the mean. Significance is denoted as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.
Fig 3.
Chronic iron overload increases the total number of lysosomes, reduces lysosome acidification, and results in lysosomal dysfunction in TDM cells.
(A) Representative images of TDM cells in the presence or absence of chronic iron overload were obtained by LysoTracker staining and confocal microscopy. (B) The intensity of LysoTracker Red fluorescence was quantified by flow cytometry. The results indicated that the number of lysosomes increased in TDMs following chronic iron exposure. (C) Representative images showed immunofluorescence staining of LAMP1 and LAMP2 in TDM cells with or without chronic iron overload. (D) The intensity of red fluorescence due to immunofluorescence-stained LAMP1 and LAMP2 was quantified by flow cytometry and the results indicated that the number of lysosomes increased in TDMs following chronic iron exposure. (E) LAMP-1, LAMP2, immature cathepsin B, cathepsin D, and TFEB proteins were significantly increased following chronic iron overload of TDM cells. The cell lysates of TDM and I-TDM cells were used for immunoblot analyses with actin as loading control. The results are shown in the right panel. Bars, mean± SEM. (F) The nuclear localization of TFEB proteins in I-TDM cells. TDM and I-TDM cells were fixed with paraformaldehyde, and the fixed cells were incubated with rabbit anti-TFEB antibodies, and with goat anti-rabbit FITC secondary antibody. Scale bars, 20 μm. (G) The lysosomal pH in TDM, I-TDM, I-TDM cells treated with DFO and I-TDMs incubated in iron-free media was quantified using LysoSensor Yellow/Blue dextran and fluorescence ELISA reader. (H) Cathepsin D activity was measured in TDM and I-TDM cells using a fluorometric cathepsin D activity assay. The data represent the average of triplicate experiments and the error bars indicate the standard error of the mean. Significance is denoted as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.
Fig 4.
Chronic iron overload disrupts the autophagic flux in TDM cells.
(A) The protein levels of LC3-II and p62 in TDM cells with or without chronic iron overload were examined by immunoblotting. (B) In the presence of 10 μM BafA1, chronic iron overload of TDM cells did not increase LC3-II and p62 levels compared with LC3-II and p62 levels in TDM cells treated with BafA1 alone. Actin served as loading control. The quantification results are shown in the right panel. Bars, mean± SEM.