Patient demographics.

<p>Patient demographics.</p

Iron uptake enhances M1 polarization.

<p>A. Human MDMs were seeded in 48 well dishes at cell density of 2×10⁶ cells/ml and polarized according to protocol. Various concentrations of FeCl₃ (0–100 µM) were added to the differentially polarized macrophages. Where indicated, cells were pre-incubated with human myelin (30 µg/ml) for 24 hrs before addition of FeCl₃. After 10 hrs, cell-free supernatants were collected and TNF-α and IL-10 concentrations were measured by ELISA. Iron uptake increased secretion of TNF-α in M1-polarized but not in M0 and M2-polarized macrophages and reduced IL-10 in M2-polarized macrophages. Cytokine secretion did not change in myelin-laden macrophages after exposure to iron. Results are expressed as means ± SEM from three separate experiments. *p<0.05; **p<0.01; ***p<0.005 by Student's t test. B. For measurement of reactive oxygen species (ROS), cells were loaded with 30 µM of oxidant-sensitive DCF-DA dye after exposure to iron and fluorescent emission was measured at 540 nm. The experiment was performed in triplicates and repeated three times. The data are expressed as change in fluorescence/8×10⁴ cells (± SEM). Iron uptake increased generation of ROS secretion in non-phagocytosing macrophages in all polarization states, most prominently in M1 polarization. Myelin-laden macrophages did not respond to iron exposure. *p<0.05; **p<0.005 by Student's t test. C. To visualize ROS generation, macrophages seeded on coverslips were incubated with 30 µM FeCl₃ for 2 hrs and subsequently loaded with 30 µM of DCF-DA. Images were acquired at timed intervals (representative images at 10 min are shown). The results recapitulate ROS quantification in (B): ROS was increased in iron-laden MDMs [d–f] compared to naïve [a–c] or myelin-laden MDMs [g–i]. In contrast, labeling for the M2 polarization marker CD206, revealed decreased expression of CD206 in macrophages exposed to iron [dd–ff] and increased expression in myelin-laden macrophages [gg–ii].</p

Antibodies.

<p>Antibodies.</p

FLAIR (3T) and GRE phase (7T) images of a patient with active relapsing-remitting MS.

<p>FLAIR images show numerous white matter MS lesions of which 2 are magnified (inset, red arrows). Phase imaging at 7T phase/GRE reveals a hypointense ring corresponding with one lesion on FLAIR. The other lesion is not visible on 7T GRE (inset, arrows).</p

Iron and myelin uptake in human monocyte-derived macrophages (MDMs).

<p>Non-polarized M0, M1- and M2-polarized macrophages were stained for neutral lipids (oil red-O) and iron (Perls). In untreated cultures, macrophages did not contain iron or significant amounts of lipids [a–c]. In macrophages exposed to FeCl₃ (10 µM; 10 hrs), Perls' staining showed polarization-dependent iron uptake [d–f]. Exposure of iron-rich macrophages to purified human myelin (30 µg/ml; 24 hrs) lead to depletion of iron from myelin-phagocytosing macrophages in all polarization states [g–i]. Presence of iron within macrophages was closely mirrored by macrophage expression of ferritin: macrophages without iron-load express little ferritin [aa–cc], while iron loading leads to increased expression of ferritin [dd–ff], particularly in M1 macrophages [ee]. Addition of myelin to iron-rich macrophages resulted in substantial reduction of ferritin in all polarization states [gg–ii]. In macrophages exposed first to myelin [j–l] and subsequently to iron, iron accumulation was significantly reduced compared to that in naive macrophages [m–o]. When M1 macrophages were incubated with myelin in the presence of the receptor-associated protein (GST-RAP), which binds to the presumed receptor for myelin ingestion, LRP-1, and inhibits interaction with other ligands, myelin ingestion was prevented [p]. Blockade of myelin ingestion in M1 macrophages with GST-RAP and subsequent exposure to FeCl₃ led to unimpeded iron uptake [q]. In contrast to myelin, internalization of fluorescent polystyrene microspheres by M1 macrophages did not prevent subsequent iron uptake [r].</p