Figure 2: Alveolar macrophage endocytosis of ITO-NPs enhances TNF-α and IL-1β pro-inflammatory cytokine production.
(a) Schematic diagram of the experimental procedures to consider alveolar macrophage response upon exposure to ITO-NPs, SiO2-NPs or MSU. (b) TEM of non-exposed MH-S cells shows preserved cytoplasmic organelles, regular microvilli protruding, and well-delineated nucleus with heterochromatin. (c) MH-S cells exposed for 6 h to 50 μg/mL ITO-NPs exhibited early endosomes containing ITO-NPs appearing as dark spots. At 24 h post-exposure to ITO-NPs at 50 μg/mL, endosomes in MH-S contained an increased amount of ITO-NPs. (b,c) Original magnification x3000, x4000 and x8000, 1 representative TEM image is shown from at least 3. (d) MH-S cell production of TNF-α upon exposure to increasing doses, i.e. 50, 100 and 500 μg/mL, of ITO-NPs. MH-S cells exposed to ITO-NPs at 500 μg/mL and examined at 3, 6, 12 and 16 h for secretion of TNF-α. (e) Intracellular TNF-α level found in MH-S cells exposed ITO-NPs compared to unstimulated MH-S. (f) Secreted and intracellular TNF-α levels in MH-S cells treated with dexamethasone (DEX) and exposed to ITO-NPs. (g) IL-10 production in MH-S cells exposed to ITO-NPs with DEX treatment and compared to controls. (h) MH-S cells treated with or without Cyto D and exposed to ITO-NPs for 12 h were analyzed in 3 representative areas (RA) on x500 TEM images. The number of endocytic cells containing ITO-NPs was compared to the total number of nucleated cells in RA; data are given as percentage of endocytic cells. Cyto D effect was evaluated for TNF-α secretion and intracellular TNF-α expression in MH-S upon exposure to ITO-NPs. (i) Intracellular IL-1β level was evaluated in MH-S cells with ITO-NP exposure; with or without DEX and Cyto D treatment compared to controls. The concentration of ITO-NPs was 500 μg/mL and time of exposure 16 h, unless stated otherwise. (b–i) n = 3–8 ± SEM, *p < 0.05. TEM abbreviations, Cyto = cytoplasm, N = nucleus, M = mitochondria and E = endosome.
