Fig 2: CD63-positive ferroptosis-dependent EVs (FedEVs) derived from macrophages simultaneously exposed to asbestos and iron in vivo and in vitro. (a) Histological images of peritoneal wall in situ in the murine model 6 months after ip injection of 3 mg crocidolite; hematoxylin and eosin (HE) image (left upper), Berlin blue staining (insoluble iron as bluish green, left lower), phase-contrast with red fluorescent image (right panels); cell-specific marker: MSLN (mesothelin), mesothelial cell; αSMA (smooth muscle actin), myofibroblast; CD68, macrophage; white arrowheads, crocidolite (bar = 100 μm). (b) High-resolution fluorescent image in granuloma; red, cell specific marker; green, CD63; blue, Hoechst33342 nuclear staining (bar = 20 μm). (c) Quantification of fluorescent intensity of CD63 in different cells surrounding granuloma (n = 30; mean ± SEM). (d) Schematic image of peritoneal wall surrounding the asbestos-induced granuloma. (e, g) Immunoblot analysis of CD63 in cells under ferroptosis and their media; HT1080 fibrosarcoma cells were exposed to RSL3 (10 μM, 24 h), and phorbol myristate acetate (PMA)-primed THP1 macrophage cells were exposed to crocidolite (15 μg/cm2) in the presence of absence of ferric ammonium citrate (Fe, 100 μg/ml) up to 96 h. (f, h) Membrane-catch transmission electronmicroscopy (TEM) images of EVs on carbon grids (bar = 200 nm). (i–l) The precision of nanoparticle tracking analysis (NTA) concentration measurements counted size-distribution of EVs derived from HT1080 (24 h) and THP1 (96 h) in different conditions in i and j. NTA concentration measurements counted number of EVs in k and l (mean ± SEM). Refer to text for details. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: Overview of image processing for multiplex immunohistochemistry (mIHC) of the cochlea. (A) Schematic view of the mIHC. The cochlea of 4–6 weeks old mice were harvested, fixed with 4% paraformaldehyde, decalcified, and embedded in paraffin. Sections were deparaffinized and immunostained with a primary antibody and visualized by using chromogenic staining (AEC; 3-amino-9-ethyl-carbazole). After imaging with a whole slide scanner, the antibody and chromogenic stains were stripped out with a serial gradient of ethyl alcohol until the signals disappeared entirely. The staining and stripping steps were repeated for six different macrophage markers (F4/80, CD68, CD206, CD86, Iba1, and CD163) and hematoxylin. (B) Image processing. The images were co-registered to align the same area in each slide, and the images were converted into pseudo-colored single-marker images to identify the positive signals of each marker. (C) Merged image of all the markers. Individual images were merged into one image showing the expression of all the markers, and analyzed to identify the markers expressed in each F4/80+ macrophage. Different areas of the cochlea were examined to identify the macrophage diversity. SG: spiral ganglia, SV: stria vascularis, M: modiolus, OC: organ of Corti, AN: auditory nerve. (D) Magnified area showing the markers expressed by the macrophages in the inner ear. Some macrophages expressed only one marker F4/80 (arrow), whereas others expressed four (double-head arrow), five (double-tail arrow), and even all the markers (arrowhead). Scale bar: the whole cochlea, 500 μm; magnified region, 30 μm.

Fig 4: Classification of mixed macrophages according to the intensity of expressed markers. (A) Watershed cell segmentation to identify the boundaries of single macrophages. Cochlea were obtained from 4-weeks old mice, and immunostained using the mIHC. The red line denotes the boundaries of each macrophage with F4/80 staining. (B) Quantification of the population in the resting state to identify the percentage of each category in the modiolus including auditory nerve and spiral ganglia. Mid-modiolar sections were analyzed, and macrophages population of each category, M0, M1-like, M2-like and M1/M2, were quantified (n = 5). The M0 non-activated macrophages represented the major percentage among all macrophages, with a significant difference compared to other categories. The mixed M1/M2 macrophages were presented minimally, while the M1-like and M2-like macrophages expressing single marker represented the least percentages. *P < 0.05, ****P < 0.0001, ns: not significant, by one-way ANOVA with Tukey's multiple comparisons test. The error bars represent standard deviation from the mean. (C) Example of mixed macrophages in which M1 markers (CD68, CD86) expression was similar to that of the M2 markers (CD206, CD163). (D) A representative macrophage of a group of mixed macrophages in which the CD86 (M1 marker) showed the highest expression compared to the other markers. (E) Illustration of another group of mixed macrophages in which CD163 (M2 marker) had the highest expression among all markers. (F) Single-cell-based marker intensity for each mixed macrophage in the modiolus including auditory nerve and spiral ganglia. Each dot represents a single mixed macrophage (n = 74 from 7 cochleae); the mean intensity of M1 and M2 markers in each macrophage are plotted against each other. The cut-off value was set at 1, below which most of the macrophages were included, and the different types of macrophages were identified. The red box marks the area of the dominant cell population in which most of the macrophages represent M1 and M2 markers equally within the cut-off value. The numbers indicate the percentages of the cell population of each quadrant; in the lower right quadrant, M1-polarized cells expressing higher M1 marker intensities (>1) than M2 cells, 8.44, 3.90, 9.49, and 5.23%: the upper left quadrant, M2-polarized cells expressing higher M2 marker intensities (>1) than M1 cells, 6.56, 3.90, 10.83, and 9.78%: the upper right quadrant, cells strongly expressing both M1 and M2 markers, 2.56, 5.23, 1.51, and 3.90%.

Fig 5: Macrophages and pro-inflammatory cytokines in adipose tissue. The presence of macrophages in para-ovary and inguinal adipose tissues was investigated by immunofluorescence microscopy using anti-CD68 antibodies (A) (scale bar = 100 μm). The macrophages numbers of CD68 staining for para-ovary (B) and inguinal (F) adipose tissue were counted. N = 3 for each group. The levels of IL-1β (C), TNF-α (D) and IL-6 (E) in para-ovary adipose tissue homogenates and the levels of IL-1β (G), TNF-α (H) and IL-6 (I) in inguinal adipose tissue homogenates were determined by ELISA. Values are expressed as mean ± SEM. ** p < 0.01, *** p < 0.001, smoke group compared with air group; N = 13–14 mice/group were used for ELISA.