Journal: JCI Insight

Article Title: MTH1 favors mesothelioma progression and mediates paracrine rescue of bystander endothelium from oxidative damage

doi: 10.1172/jci.insight.134885

Figure Lengend Snippet: (A) AE17 and AE17 MTH1–overexpressing cells secrete large genomic DNA fragments. Nucleic acid isolated from AE17 and AE17 MTH1–overexpressing (AE17mth1over) cell culture supernatants were analyzed by capillary electrophoresis. Representative electropherograms of AE17 (top) and AE17mth1overexpressing cell (bottom) nucleic acids. (B) Oxidative state of cfDNA secreted by AE17 tumor cells is higher than that of AE17 mth1–overexpressing cells. Addition of antioxidant can prevent 8-Oxo-dG incorporation into DNA. cfDNA isolated from cell culture supernatants of AE17 (n = 4), AE17mth1over (n = 3), or AE17 cells treated with NAC (5 mM, overnight) (n = 3) was analyzed for the presence of 8-Oxo-dG by ELISA. Results were normalized to total DNA (ng). (C) cfDNA secreted by AE17 cells activates NF-κB in TECs. Serum-starved TECs were treated with vehicle or cfDNA from AE17 for 4 hours. Phosphorylated and total p65–NF-κB was detected by Western blot. (D) “More oxidized” cfDNA triggers a higher NF-κB activation than “less oxidized” cfDNA, through TLR9. Serum-starved TECs were treated with TLR9i (2 μg/mL) or vehicle for 40 minutes and subsequently treated with 20 ng/mL cfDNA from AE17, AE17 MTH1–overexpressing cells, or AE17 cells treated with NAC for 4 hours. Phosphorylated and total p65–NF-κB was measured by Western blot. (E) cfDNA of MTH1-overexpressing tumor cells upregulates MTH1 of TECs through TLR9. TECs were treated as described in D and analyzed for MTH1 expression by Western blot. (F) NF-κB binds to the endogenous MTH1 promoter of TECs. TECs were treated as C (n = 3). TNF-α (20 ng/mL) was used as a positive control. Binding of NF-κB to MTH1 gene promoter was determined by ChIP assay and Real-time PCR. Results were normalized to the input DNA control. A negative control (NC) (no antibody) was included. (G) TECs were treated as in C, and apoptotic cells were determined upon annexin V–PI staining (AE17, n = 7; AE17mth1over, n = 7; AE17+NAC, n = 4). (C–E) One representative blot of 3 independent experiments. All data are presented as the mean ± SEM. (B, F, G) *P < 0.05 compared with indicated groups by 1-way ANOVA (with Bonferroni’s post hoc test for multiple comparisons). (C, D, E) *P < 0.05 compared with indicated groups by 2-tailed Students’ t test.

Article Snippet: TLR9 inhibitor chloroquine (Merck) was used at 2 μg/mL 40 minutes before addition of cfDNA.

Techniques: Isolation, Cell Culture, Electrophoresis, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay, Expressing, Positive Control, Binding Assay, Real-time Polymerase Chain Reaction, Negative Control, Staining

Journal: Scientific Reports

Article Title: Toll-like receptor 9-positive plasmacytoid dendritic cells promote Th17 immune responses in oral lichen planus stimulated by epithelium-derived cathepsin K

doi: 10.1038/s41598-023-46090-3

Figure Lengend Snippet: Identification and distribution of TLR9-expressing cells in BM specimens from patients with OLP. ( A ) Representative images of immunofluorescence staining of TLR9 (red) in BM specimens from patients with OLP and HK. Scale bars, 100 μm. ( B ) Number of TLR9-positive cells in BM specimens from patients with OLP (n = 20) and HK (n = 12). ( C ) Representative images of double immunofluorescence staining for TLR9 (red), CD20 (blue), and CD123 (green) in BM specimens from patients with OLP. Scale bars, 100 μm. ( D ) Number of TLR9-expressing CD20- and CD123-positive cells in BM specimens from patients with OLP (n = 20). In ( B ) and ( D ), data are shown as box plots. Each box represents the upper and lower interquartile range. Lines inside the boxes represent the median. Symbols represent individual subjects. *** P < 0.001, **** P < 0.0001, by one-way ANOVA ( B ) or Mann–Whitney U tests ( D ). See Fig. for other definitions.

Article Snippet: Cells were then stimulated with 3 μM of the TLR9 agonist CpG DNA (Catalog No. HC4037; Hycult Biotechnology, Uden, The Netherlands) and/or 400 nM of CTSK (Catalog No. SRP6561-5UG; Sigma-Aldrich, Saint Louis, MO, USA) and/or 10 μM of the TLR9 inhibitor iODN (Catalog No. IAX-200-050; Innaxon, Tewkesbury, UK) for 2 days.

Techniques: Expressing, Immunofluorescence, Staining, Double Immunofluorescence Staining, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Toll-like receptor 9-positive plasmacytoid dendritic cells promote Th17 immune responses in oral lichen planus stimulated by epithelium-derived cathepsin K

doi: 10.1038/s41598-023-46090-3

Figure Lengend Snippet: Association of CD123 + plasmacytoid dendritic cells (pDCs) with Th17 cell differentiation. ( A ) Schematic illustration of the experimental procedures; human CD123 + pDCs were extracted from peripheral blood mononuclear cells (PBMCs) and then stimulated by the Toll-like receptor 9 (TLR9) agonist CpG DNA, TLR9 inhibitor iODN, and/or CTSK in vitro. ( B ) Production of Th17-related cytokines (IL-6, IL-23, and TGF-β) in CD123 + pDCs stimulated with CpG DNA, iODN, and/or CTSK (n = 3/sample). Bars show the mean ± SD. Symbols represent individual subjects. * P < 0.05, ** P < 0.01, by one-way ANOVA. ( C ) Representative images of immunofluorescence staining for CD4 (green), RORγt (pink) and DAPI (blue) in BM specimens from patients with OLP and HK. Scale bars, 100 μm. ( D ), ( E ) Quantification of CD4 + RORγt + Th17 cells in BM specimens from patients with OLP (n = 20) and HK (n = 12). ( F ) Correlation among the numbers of CTSK + cells, TLR9 + CD123 + pDCs, and CD4 + RORγt + Th17 cells in BM specimens from patients with OLP (n = 20). The correlation coefficients were determined by Spearman’s rank correlations. ( G ) Number of TLR9 + CD123 + pDCs and CD4 + RORγt + Th17 cells in BM specimens from patients with reticular type OLP (n = 9) and erosive type OLP (n = 11). In (D ), ( E ) and ( G ) data are shown as box plots. Each box represents the upper and lower interquartile range. Lines inside the boxes represent the median. Symbols represent individual subjects. ** P < 0.01, *** P < 0.001, **** P < 0.0001 by one-way ANOVA ( D and E ) or Mann–Whitney U tests ( G ). See Fig. for other definitions.

Article Snippet: Cells were then stimulated with 3 μM of the TLR9 agonist CpG DNA (Catalog No. HC4037; Hycult Biotechnology, Uden, The Netherlands) and/or 400 nM of CTSK (Catalog No. SRP6561-5UG; Sigma-Aldrich, Saint Louis, MO, USA) and/or 10 μM of the TLR9 inhibitor iODN (Catalog No. IAX-200-050; Innaxon, Tewkesbury, UK) for 2 days.

Techniques: Cell Differentiation, In Vitro, Immunofluorescence, Staining, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Toll-like receptor 9-positive plasmacytoid dendritic cells promote Th17 immune responses in oral lichen planus stimulated by epithelium-derived cathepsin K

doi: 10.1038/s41598-023-46090-3

Figure Lengend Snippet: Single cell RNA-sequencing (scRNA-seq) analysis in BM specimens from patients with OLP. ( A ) Uniform manifold approximation and projection (UMAP) of CD45 + immune cells colored by cell types. ( B ) UMAP colored by TLR9 expression. ( C ) The violin plots showing the expression pattern of the selected cell markers in each of the clusters. ( D ) The percentage of CD45 + immune cells in TLR9 + cells. ( E ) KEGG pathways of upregulated DEGs in TLR9 + pDC compared with TLR9 − pDC. ( F ) GO analysis of DEGs upregulated in TLR9 + pDC compared with TLR9 − pDC.

Article Snippet: Cells were then stimulated with 3 μM of the TLR9 agonist CpG DNA (Catalog No. HC4037; Hycult Biotechnology, Uden, The Netherlands) and/or 400 nM of CTSK (Catalog No. SRP6561-5UG; Sigma-Aldrich, Saint Louis, MO, USA) and/or 10 μM of the TLR9 inhibitor iODN (Catalog No. IAX-200-050; Innaxon, Tewkesbury, UK) for 2 days.

Techniques: RNA Sequencing Assay, Expressing

Journal: Scientific Reports

Article Title: Toll-like receptor 9-positive plasmacytoid dendritic cells promote Th17 immune responses in oral lichen planus stimulated by epithelium-derived cathepsin K

doi: 10.1038/s41598-023-46090-3

Figure Lengend Snippet: Schematic model of CTSK-induced TLR9 signaling in pDCs, leading to inflammation in OLP. TLR9 expressed on CD123-positive pDCs in subepithelial layers recognizes viral DNA. CTSK released from the LE activates TLR9 signaling in pDCs and their production of Th17-related cytokines, including IL-6, IL-23, and TGFβ, which leads to Th17-associated inflammation in OLP. See Fig. for other definitions.

Article Snippet: Cells were then stimulated with 3 μM of the TLR9 agonist CpG DNA (Catalog No. HC4037; Hycult Biotechnology, Uden, The Netherlands) and/or 400 nM of CTSK (Catalog No. SRP6561-5UG; Sigma-Aldrich, Saint Louis, MO, USA) and/or 10 μM of the TLR9 inhibitor iODN (Catalog No. IAX-200-050; Innaxon, Tewkesbury, UK) for 2 days.

Techniques: