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. Author manuscript; available in PMC: 2020 Oct 1.

Published in final edited form as: Small. 2019 Aug 28;15(42):e1901642. doi: 10.1002/smll.201901642

Figure 4. — (A) Schematic representation to demonstrate how injury to the lysosomal membrane by nanocellulose materials could induce cathepsin B release, and NLRP3 inflammasome activation. (B) Confocal microscopy demonstrating the effects of nanocellulose on cathepsin B release in THP-1 cells. Cathepsin B release from damaged lysosomes was identified by Magic Red staining. THP-1 cells were incubated with 300 μg/mL nanocellulose for 24 h. After fixation and permeabilization, cells were stained with Magic Red (ImmunoChemistry Technologies) and Hoechst 33342 dye, followed by visualization under a confocal 1P/FCS inverted microscope. MSU crystals at 100 μg/mL were used as a positive HAR control material that induces lysosomal damage. This leads to diffuse intracellular release of the red fluorescent dye compared to the punctate staining pattern of intact lysosomes in control cells. (C) Dose-dependent IL-1β production in THP-1 cells exposed to nanocelluloses. THP-1 cells were exposed to 37.5, 75, 150, and 300 μg/mL of each of the cellulose suspensions for 24 h. IL-1β levels were determined in the supernatants collected by ELISA. *p < 0.05 compared to control. (D) Assessment of IL-1β production by nanocelluloses at 300 μg/mL using ELISA in the supernatants of the cells shown in Figure 4C. (E) The involvement of the NLRP3 inflammasome was confirmed by using NLRP3 deficient (NLRP3^−/−) and ASC deficient (ASC^−/−) THP-1 cells. (E) The cathepsin B inhibitor, CA-074 methyl ester, and (F) the caspase 1 inhibitor, z-YVAD-fmk, were used to confirm the involvement of cathepsin B and caspase-1 in nanocellulose-triggered inflammasome activation. *p<0.05 compared to the response in THP-1 cells in the absence of the inhibitors; # p < 0.05 for pairwise comparisons as shown.

Figure 4. — (A) Schematic representation to demonstrate how injury to the lysosomal membrane by nanocellulose materials could induce cathepsin B release, and NLRP3 inflammasome activation. (B) Confocal microscopy demonstrating the effects of nanocellulose on cathepsin B release in THP-1 cells. Cathepsin B release from damaged lysosomes was identified by Magic Red staining. THP-1 cells were incubated with 300 μg/mL nanocellulose for 24 h. After fixation and permeabilization, cells were stained with Magic Red (ImmunoChemistry Technologies) and Hoechst 33342 dye, followed by visualization under a confocal 1P/FCS inverted microscope. MSU crystals at 100 μg/mL were used as a positive HAR control material that induces lysosomal damage. This leads to diffuse intracellular release of the red fluorescent dye compared to the punctate staining pattern of intact lysosomes in control cells. (C) Dose-dependent IL-1β production in THP-1 cells exposed to nanocelluloses. THP-1 cells were exposed to 37.5, 75, 150, and 300 μg/mL of each of the cellulose suspensions for 24 h. IL-1β levels were determined in the supernatants collected by ELISA. *p < 0.05 compared to control. (D) Assessment of IL-1β production by nanocelluloses at 300 μg/mL using ELISA in the supernatants of the cells shown in Figure 4C. (E) The involvement of the NLRP3 inflammasome was confirmed by using NLRP3 deficient (NLRP3^−/−) and ASC deficient (ASC^−/−) THP-1 cells. (E) The cathepsin B inhibitor, CA-074 methyl ester, and (F) the caspase 1 inhibitor, z-YVAD-fmk, were used to confirm the involvement of cathepsin B and caspase-1 in nanocellulose-triggered inflammasome activation. *p<0.05 compared to the response in THP-1 cells in the absence of the inhibitors; # p < 0.05 for pairwise comparisons as shown.