Journal: Particle and Fibre Toxicology

Article Title: Zinc oxide nanoparticles exacerbate skin epithelial cell damage by upregulating pro-inflammatory cytokines and exosome secretion in M1 macrophages following UVB irradiation-induced skin injury

doi: 10.1186/s12989-024-00571-z

Figure Lengend Snippet: ZnONPs induced cytotoxicity and innate immune responses through Toll-like receptor (TLR) signaling in THP-1-derived macrophages. THP-1 cells were incubated for 24 h in the presence of 10 ng/ml PMA and then exposed to various concentrations of ZnONPs for different times. A Effects of ZnONPs on THP-1-derived macrophage viability after 24 or 48 h. Cell viability was measured by MTT assays. B ZnONP-induced ROS generation in THP-1-derived macrophages. After being exposed to ZnONPs, the cells were incubated with 10 μM DCFH-DA for 30 min to stain H 2 O 2 and analyzed by flow cytometry. Histograms show the percentages of DCFH-DA fluorescence compared with the control. The data are presented as the mean ± SD from three independent experiments. (* p < 0.05, compared with control) C THP-1-derived macrophages were treated with ZnONPs (10 and 15 μg/ml) for 18 h, and the mRNA expression of TLR1, TLR3 and TLR6 was examined by RT‒qPCR and normalized to GAPDH expression. The data were presented as the mean ± SD from three independent experiments. The results showed that ZnONPs significantly triggered TLR1 and TLR3 activation. D THP-1-derived macrophages were treated with ZnONPs (10 μg/ml) for various times, and NFκB (p-p65) protein expression was analyzed by western blotting. E The mRNA levels of the proinflammatory cytokines TNF-α and IL-1β were determined by RT‒qPCR and normalized to GAPDH expression. The data are presented as the mean ± SD from three independent experiments. ZnONPs increased TNF-α and IL-1β mRNA expression at 18 h. (* p < 0.05, ** p < 0.01 compared with control). F THP-1-derived macrophages were treated with ZnONPs (10 μg/ml), and CU-CPT22 or CU-CPT 4a for 16 h, then NFκB (p-p65) protein expression was analyzed by western blotting. CU-CPT22 and CU-CPT 4a are inhibitors specific to TLR1/2 and TLR3, respectively. G Cells were treated with ZnONPs (10 μg/ml), and CU-CPT22 (50 μM) or CU-CPT 4a (30 μM) for 24 h. TNF-α and IL-1β protein levels in the culture medium were measured by ELISA. The data were presented as the mean ± SD from three independent experiments. (* p < 0.05, ** p < 0.01 compared with control, # p < 0.05, ## p < 0.01 compared with ZnONPs alone)

Article Snippet: CU-CPT22 and CU-CPT4a were purchased from MedChemExpress (NJ, USA).

Techniques: Derivative Assay, Incubation, Staining, Flow Cytometry, Fluorescence, Control, Expressing, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: Lysosomal exocytosis of HSP70 stimulates monocytic BMP6 expression in Sjögren’s syndrome

doi: 10.1172/JCI152780

Figure Lengend Snippet: ( A ) Venn diagram showing increased expression of potential ligands for TLR4 (listed inside the largest oval) in minor salivary glands (yellow oval), saliva (blue oval), and/or serum/plasma (shown in red) from patients with SS. ( B ) Serum HSP70 and BMP6 levels in patients with SS ( n = 50) and HVs ( n = 30). Boxes represent first, second, and third quartiles. †† P < 0.01, by Wilcoxon test. RFU, relative fluorescence units. ( C and D ) THP1 cells were treated with the indicated recombinant proteins at the indicated concentration for 4 hours. C, control. ( E ) THP1 cells were treated with boiled HSP70 (1 μg/mL) or LPS (100 ng/mL) for 4 hours. ( F ) THP1 cells were pretreated with TLR1/2 antagonist (CUCPT22, 20 μM) or TAK242 (40 μM) 1 hour prior to HSP70 simulation. ( G ) WT, MYD88 –/– , or IRF3 –/– THP1 cells were stimulated with HSP70 for 4 hours. TNFA and BMP6 transcript levels in the cells were quantified after each treatment using the ΔΔCt method relative to ACTB expression. Values shown are mean ± SEM of 3 independent experiments. ** P < 0.01, by Student’s t test with multiple testing correction using Dunnett’s method ( C – G ).

Article Snippet: The following chemical reagents and recombinant proteins were purchased: LPS, ZDEVD, YVAD, and vacuolin-1 (MilliporeSigma); loxoribine, 3M002, and ODN2216 (InvivoGen); ZVAD and HSP60, -70, and -90 (Enzo Life Sciences); CUCPT22 and TAK242 (Tocris Bioscience); IFN-γ and HMGB1 (R&D Systems); and mouse HSP70-neutralizing antibody (Thermo Fisher Scientific, MA3-009).

Techniques: Expressing, Clinical Proteomics, Fluorescence, Recombinant, Concentration Assay, Control

Journal: The Journal of Clinical Investigation

Article Title: Lysosomal exocytosis of HSP70 stimulates monocytic BMP6 expression in Sjögren’s syndrome

doi: 10.1172/JCI152780

Figure Lengend Snippet: ( A ) Scatter plot showing enriched gene ontology in patients with SS with high BMP6 expression compared with those with normal BMP6 expression. ( B ) Correlation between LAMP3 and BMP6 transcript expression in minor salivary glands of patients with SS ( n = 43). ( C ) Schematic showing the methods used in the in vitro assays. ( D ) Representative Western blot with the indicated antibodies using lysates of salivary acinar or ductal cells 72 hours after transfection with empty and/or LAMP3 expression plasmids. ( E ) HSP70 concentration in culture supernatant collected 96 hours after transfection. ( F ) THP1 cells were treated with the culture supernatant of acinar or ductal cells with or without CUCPT22 (20 μM) or TAK242 (40 μM). BMP6 transcript levels in THP1 cells were evaluated 20 hours after stimulation using the ΔΔCt method relative to ACTB . Values shown are the mean ± SEM of 3 independent experiments. ** P < 0.01, by Student’s t test with multiple testing correction using Tukey’s method ( E and F ).

Article Snippet: The following chemical reagents and recombinant proteins were purchased: LPS, ZDEVD, YVAD, and vacuolin-1 (MilliporeSigma); loxoribine, 3M002, and ODN2216 (InvivoGen); ZVAD and HSP60, -70, and -90 (Enzo Life Sciences); CUCPT22 and TAK242 (Tocris Bioscience); IFN-γ and HMGB1 (R&D Systems); and mouse HSP70-neutralizing antibody (Thermo Fisher Scientific, MA3-009).

Techniques: Expressing, In Vitro, Western Blot, Transfection, Concentration Assay

Journal: GeroScience

Article Title: Contribution of Porphyromonas gingivalis lipopolysaccharide to experimental periodontitis in relation to aging

doi: 10.1007/s11357-020-00258-1

Figure Lengend Snippet: Age-dependent expressions of Pg-LPS receptors, TLR2, and TLR4, on the surface of CD11b+CSF1r-eGFP+ OCPs. Osteoclast precursors (OCPs) were FACS sorted from the spleen of young (2 months old) and aged (24 months old) CSF1r-eGFP-KI mice and then were stimulated with macrophage colony-stimulating factor (M-CSF) (30 ng/ml) and RANKL (5 ng/ml). After 24 h of stimulation, the expression of TLR2 (a and b) and TLR4 (c and d) was evaluated by multicolor flow cytometry. ***p < 0.001

Article Snippet: FACS-sorted CD11b+CSF1r-eGFP+ OCPs were seeded in 96-well plates at a density of 1 × 10 4 cells/well in α-MEM medium (Life Technologies) containing 10% FBS (Atlanta Biologicals) and 30 ng/ml of M-CSF and 5 ng/ml of RANKL (BioLegend) recombinant proteins in the presence or absence of various concentrations of ultrapure Pg -LPS (1, or 10 ng/ml; InvivoGen), CU-CPT22 (1 and 10 μM; TLR2 antagonist; Cayman Chemical), and TAK242 (1 and 10 μM; TLR4 antagonist; Cayman Chemical).

Techniques: Expressing, Flow Cytometry

Journal: GeroScience

Article Title: Contribution of Porphyromonas gingivalis lipopolysaccharide to experimental periodontitis in relation to aging

doi: 10.1007/s11357-020-00258-1

Figure Lengend Snippet: The effects of Pg-LPS and TLR’s antagonists on RANKL-induced osteoclastogenesis in relation to aging. Microscopic evaluation (a) of the TRAP staining and quantification of the number of TRAP+ multinucleated cells (b) in the RANKL-stimulated young and aged OCPs exposed to Pg-LPS in vitro. Effects of TLR4 (CAS 243984-11-4) and TLR2 antagonists (CU-CPT22) on young (c) and aged (d) RANKL-stimulated OCPs exposed to Pg-LPS. OCPs were FACS sorted from the spleen of young and aged mice and then were stimulated with RANKL in the presence or absence of various concentrations of Pg-LPS as well as TLR2 and TLR4 antagonists. The cells were evaluated at day 6 after stimulation. **p < 0.01, ***p < 0.001

Article Snippet: FACS-sorted CD11b+CSF1r-eGFP+ OCPs were seeded in 96-well plates at a density of 1 × 10 4 cells/well in α-MEM medium (Life Technologies) containing 10% FBS (Atlanta Biologicals) and 30 ng/ml of M-CSF and 5 ng/ml of RANKL (BioLegend) recombinant proteins in the presence or absence of various concentrations of ultrapure Pg -LPS (1, or 10 ng/ml; InvivoGen), CU-CPT22 (1 and 10 μM; TLR2 antagonist; Cayman Chemical), and TAK242 (1 and 10 μM; TLR4 antagonist; Cayman Chemical).

Techniques: Staining, In Vitro