Chloroquine Interference with Hemoglobin Endocytic Trafficking Suppresses Adaptive Heme and Iron Homeostasis in Macrophages: The Paradox of an Antimalarial Agent
Figure 5
Chloroquine impairs Hb:Hp trafficking through the endosomal-lysosomal compartment. (a) CD163-positive HEK cells incubated 15 min after endocytosis with Alexa 594-labeled Hb:Hp complexes and Alexa 488-labeled transferrin, with and without chloroquine pretreatment. The proportion of endocytosed Hb:Hp complexes colocalizing with the early endosomal compartment (transferrin-positive) increases substantially after chloroquine exposure. (b) CD163-positive HEK cells incubated for 15 min (upper two rows) or 40 min (lower two rows) after endocytosis with Alexa 594-labeled Hb:Hp complexes. The lysosomal compartment is stained with anti-LAMP-1 antibody and Alexa 488-labeled secondary antibody. Early colocalization of Hb:Hp complexes in the lysosomal (LAMP-1) compartment after 15 min is considerably reduced by pretreatment with chloroquine. In contrast, after 40 min, chloroquine pretreatment leads to marked accumulation of Hb:Hp complexes in the lysosomes. Optical magnification of all images is 630x. DAPI-stained nuclei are blue, and scale bars are spaced at 5 μm. The percentage of colocalization is shown in the graphs on the right with black bars indicating control cells and gray bars indicating chloroquine-treated cells; -5 images per condition that were recorded on independent samples.
