Figure 1.
Clofazimine inclusions formed in macrophage-like cells in vivo.
(A) Mice fed with clofazimine (above) showed reddish pigmentation visible in the ear, tail, and skin when compared to mice treated with vehicle only (below). (B) Weight gains in mice fed with and without clofazimine were comparable (N = 40, ▪, vehicle; ○, treated; *, P<0.01, end-point T-test). (C) Biochemical analysis of various organs revealed differences in the accumulation and retention of clofazimine after wash out (*, P<0.01, N = 5 per group, ANOVA). (D) Ruby red inclusions appeared in frozen sections of spleen, lung and liver, but not in kidneys of 8 wk supplemented diet. H, hepatocyte; V, blood vessel; M, microgranulomas. (E) Intracellular inclusions were extracted in perfusion-fixed liver upon ethanol-dehydration and staining with toluidine blue. Arrows indicate needle-like cavities remaining after extraction. (F) Histological sections revealed cellular changes in liver of mice fed with clofazimine. H&E staining, F4/80 macrophage specific marker, Masson's trichrome staining (MTS, collagen fibers), von Willebrand factor (vWF, endothelium) and alpha smooth muscle actin (αSMA). K, Kupffer cells. Scale bar = 10 µm unless otherwise indicated.
Figure 2.
Macrophages containing intracellular CLDIs were collected, plated and studied in vitro.
(A) Bone marrow macrophage (BMM) and thioglycollate elicited peritoneal macrophages (PM) were obtained from mice fed with clofazimine, attached and spread on tissue culture plastic, and were stained with Hoechst 33342 to show nuclei. (B) Peritoneal macrophages with CLDIs migrated away from large clusters when plated on tissue culture dishes. (C) Illuminating peritoneal macrophages with blue (490 nm) light triggers clofazimine release (observed in TRITC channel) from CLDIs. (D) Once removed from cells, extracellular CLDIs grew in size and became irregular in morphology, unlike intracellular CLDIs. Red blood cells (d = 8 µm) in the background serve as size markers, for reference. (E) CLDIs inside bone marrow-derived cells in suspension, stained with Trypan Blue. Scale bars = 10 µm unless otherwise indicated.
Figure 3.
CLDIs exhibited different chemical and physical properties from pure clofazimine crystals.
Polarized light and epifluorescence microscopy (using the eGFP or Cy3 fluorescence channels) showed that pure clofazimine crystals (control, A) were unchanged by different treatments. These crystals appeared birefringent and fluoresced in the standard eGFP and Cy3 channels of the epifluorescence microscope. (B) Isolated CLDIs remained intact in isotonic solution of 10% sucrose in water, did not fluoresce in the eGFP channel but fluoresced in the Cy3 channel. (C) Isolated CLDIs burst and aggregated in distilled water, and became fluorescent in the eGFP channel. (D) After exposure to 1N NaOH, isolated CLDIs partially disintegrated in different parts. Arrows point to the tips of a CLDI that were fluorescent in the eGFP channel. (E) After 15 min at 100°C, CLDIs fragmented and changed to a pale orange color. (F) CLDIs appeared to remain partly intact when viewed after 30 min sonication and 1 hour trypsin treatment. Scale bars = 10 µm. (G) Powder X-ray diffractogram for isolated CLDI and 8 wk treated mouse spleen homogenate showed a single peak at 2-theta = 7.2°Control spleen homogenate from vehicle-only treated mouse did not show this peak. As a reference, pure, solid clofazimine crystals (monoclinic and triclinic) showed many peaks at higher angle indicative of a three dimensional, molecular lattice organization.
Figure 4.
TEM and deep-etch freeze fracture electron microscopy analysis of CLDIs.
(A) Intact CLDIs were observed in the cells of the lamina propria of 8.5 wk treated mouse jejunum using TEM. P, extracted polyhedral cavities. M, mitochondria. (B) CLDIs were delimited by a lipid double layer. (C) CLDIs appeared to form from heterogeneous granular domains transforming into a lamellar structure, observed in 4.5 wk treated jejunum (D). (E) Zoomed image of the CLDI from (A) showed the lattice-like lamellar structure. (F) and (G), Discrete Fourier Transforms confirmed the regular, periodic structure of CLDIs. (H) Transversal cross section of CLDIs from 8.5 wk jejunum showed an internal organization of parallel bands and some amorphous regions indicated by (*). (I) Zoomed image revealed the trilayer membrane of 10 nm in width separated by inter-laminar space continuous with amorphous region. (J) Zoomed image of rectangle in (H), showing trilayer membrane structure comprised of a central dark band (arrows) flanked by a pair of less prominent, dark bands (triangles) on either side, separated by clear 5 nm space. The trilayer membranes were regularly spaced and formed planar stacks, with the central bands exhibiting the 18 nm spacing in the Discrete Fourier Transform (K).
Figure 5.
Deep-etch freeze-fracture microscopy of the intracellular CLDI from 6 wk treated mouse liver.
Note the outer membrane of the CLDI is studded with globular protein-like features (white triangle). The CLDI was broken during freeze-fracture, revealing a lamellar internal structure at the top.
Figure 6.
Outer membrane and internal organization of a CLDI from 6 wk treated mouse liver.
(A) Zoomed out image, revealing the exposed, inner multilamellar core with the outer double membrane layers peeled back towards the cytoplasm of the cell. The CLDI was broken open during the sample preparation process. Regions of interest (corresponding to panels B to F) are marked with letters. (B) Zoomed image of the inner lamellar surface. (C) Zoomed image of the multilamellar core region, showing variable spacings in the order of ranging from 6 to 14 nanometers. (D) Zoomed image of the innermost face of the inner bounding double membrane without the globular, protein-like features. (E) Zoomed image of the innermost face of the outer bounding double membrane. Protein-sized features are observed on the inner surface of the outer membrane (arrowheads). (F) Zoomed image of the outer double membrane. Note the large space between the double membranes, with “pillars” bridging the membranes (arrows).
Figure 7.
Deep-etch freeze-fracture electron microscopy of isolated CLDIs.
(A) Pure isolated CLDIs stood out from surrounding ice and cytosolic debris based on their elongated polyhedral shape and internal layered structure. (B) Isolated CLDIs clearly lacked the outer double membrane covering. (C) Biochemically-isolated CLDI often showed outer layers of material that appeared to be peeling off from the structure.