Journal: Cell death & disease

Article Title: Neutrophil extracellular traps impair intestinal barrier functions in sepsis by regulating TLR9-mediated endoplasmic reticulum stress pathway.

doi: 10.1038/s41419-021-03896-1

Figure Lengend Snippet: Fig. 7 TLR9 antagonist inhibits NETs-induced ER stress activation and intestinal epithelial cell injury. A The morphology of the Caco2 monolayer after NETs (300 ng/ml) treatment with or without costimulation with a TLR2 antagonist (C29), TLR4 antagonist (TAK-242), and TLR9 antagonist (ODN2088). Scale bars = 100 μm. B Cell viability tested by the cell counting kit-8. C The impact of TLR9 antagonist (ODN2088) on the protein level changes in the Caco2 epithelial barrier model was assessed by western blotting. D The intensity was quantified using ImageJ software from three independent analyses. Data are expressed as the mean ± SD. *P < 0.05, **P < 0.01.

Article Snippet: Caco2 cells were preincubated with or without various inhibitors for 1.5 h, such as ER stress inhibitor (4-PBA, 5 μM, HY-A0281, MedChem Express), TLR2 inhibitor (C29, 50 μM, HY-100461, MedChem Express), TLR4 inhibitor (TAK-242, 1 μM, HY-11109, MedChem Express), TLR9 inhibitor (ODN2088, 5 μM, tlrl-2088, InvivoGen), or NAC (10mM, A7250, SigmaAldrich).

Techniques: Activation Assay, Cell Counting, Western Blot, Software

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: Screening of a receptor target for SAA1 in HSCs (A and B) LX-2 and PRHSCs were stimulated with rhSAA1 or rmSAA1, respectively, for 24 hr, and mRNA levels of TLR2, TLR4, FPR2, RAGE, and SR-B1 were determined by RT-qPCR. (C and D) Representative western blot analysis of receptors TLR2, TLR4, FPR2, RAGE, and SR-B1 after rhSAA1 and rmSAA1 treatment of LX-2 and PRHSCs, respectively. (E and F) LX-2 and PRHSCs were transfected with reporter plasmid for NF-κB. Twenty four hr later, the medium was changed and cells were pretreated with inhibitors of TLR2 (CU-CPT22, 1 μM), RAGE (FPS-ZM1, 0.5 μM), TLR4 (TAK 242, 5 nM), and FPR2 (WRW4, 0.25 μM) and then stimulated with rhSAA1 and rmSAA1, respectively, diluted in serum-free DMEM. Twelve hr later, luciferase activity was measured in fold induction after normalization with Renilla luciferase (Rluc) control. (G and H) Co-immunoprecipitation (co-IP) experiment showing the interaction between SAA1 and TLR2 determined by silver staining and western blot analysis. (For detailed information please see section). (n = 3). Where applicable, data represent mean ± SEM ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 and (n = 3).

Article Snippet: For in vivo inhibition of TLR2 in mice liver, C57BL/6 mice were intraperitoneally injected CU-CPT22, a TLR2 antagonist (Selleck.cn).

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Control, Immunoprecipitation, Co-Immunoprecipitation Assay, Silver Staining

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: TLR2 serves as a chemotactic receptor for SAA1 and mediates migration of HSCs (A) Schematic of the TLR2 gene knockout strategy. The Cas9/sgRNA(s) target site(s) are indicated in red and were confirmed by sequencing. (B) Sequencing result of targeted region. (C) Confirmation of TLR2 gene KO at protein level determined by western blot. (D and E) WT and TLR2 −/− LX-2 cells (D) and WT and siPRHSCs (E) were transfected with reporter plasmid for NF-κB. Luciferase activity was measured as fold induction in comparison to untreated control (See also section for detail information). (F and G) Representative images showing agarose spot and Transwell migration assays of SAA1-treated WT LX-2, TLR2 −/− LX-2 cells, and non-treated control. (H and I) Representative images for immunostaining of TLR2 (H) and α-SMA + cells (I) in CU-CPT22- or VL-treated samples as shown in CCl 4 and CI injury models. Bar graph represents quantification of IHC images per 5 field(s). (J and K) Immunofluorescence images showing co-localization of SAA1 and HSCs in CU-CPT22-treated and control (VL) samples in CCl 4 (J) and CI injury models (K). (Scale bar represents 100 μm for CCl 4 injury, 200 μm for CI injury, and 50 μm for inset). Data represent mean ± SEM ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.0001 (n = 3). (For detailed TLR2 knockout strategy, see section).

Article Snippet: For in vivo inhibition of TLR2 in mice liver, C57BL/6 mice were intraperitoneally injected CU-CPT22, a TLR2 antagonist (Selleck.cn).

Techniques: Migration, Gene Knockout, Sequencing, Western Blot, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Comparison, Control, Immunostaining, Immunofluorescence, Knock-Out

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: SAA1/TLR2 axis guides homing of transplanted LX-2 toward injury site(s) (A) Schematic representation of experimental design. (B) Bioluminescence imaging of transplanted cells entrapped to the liver 24 hr after transplantation in CCl 4 injury model, (A) represents sham operated mice, (B) represents SAA1siRNA2-treated mice transplanted with WT LX-2, (C) represents Neg-siRNA-treated mice transplanted with TLR2 −/− LX-2, and (D) represents control mice transplanted with WT LX-2 cells. (C and D) Representative confocal immunofluorescence images showing the recruitment of transplanted WT and TLR2 −/− LX-2 cells at injury locus after CCl 4 and CI injuries induced in SAA1-siRNA2, Neg-siRNA, and control samples, respectively. Bar graph represents quantification of relative number of homed cells at injury site(s). (Scale bar represents 100 μM). Data represent mean ± SEM ∗p < 0.01, ∗∗p < 0.01, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001(n = 3).

Article Snippet: For in vivo inhibition of TLR2 in mice liver, C57BL/6 mice were intraperitoneally injected CU-CPT22, a TLR2 antagonist (Selleck.cn).

Techniques: Imaging, Transplantation Assay, Control, Immunofluorescence

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: SAA1/TLR2 axis induces Rac GTPase-mediated actin reorganization and migration of HSCs (A) Time course of GTPase-Rac1 activation(s) in LX-2 cells at indicated time interval (B and C) Basal and stimulated levels of GTPase-Rac1 in WT and TLR2 −/− cells as determined by pull-down assays (D and E) WT and TLR2 −/− LX-2 cells were pretreated with NSC 23766 (50 μΜ), and activation of GTP-Rac1 was determined by pull-down assay (D), and MLCK and p-MLC at Ser19 activity was determined by western blotting (E). (F and G) Migration assays showing pretreatment of the cells with NSC 23766 (50 μΜ) attenuated their migration(s) in agarose spot (F) and Transwell (G) assays. (H) Time course of PI3K activations after treatment of LX-2 cells at indicated time points. (I and J) WT and TLR2 −/− cells were pretreated with LY294002 (10 μΜ), and the phosphorylation of PI3K at p85 subunits was determined by western blot analysis, and the activations of Rac GTPase were determined by pull-down assay. (K and L) Migration assays showing pretreatment of the cells with LY294002 attenuated their migration(s) in agarose spot (K) and Transwell (L) assays. Photographs are representatives of (n = 6). (Scale bar represents 200 μm). Data represent mean ± SEM ∗p < 0.01 and ∗∗p < 0.001 (n = 3). Photographs are representatives of (n = 6). (Scale bar represents 200 μm). Data represent mean ± SEM ∗p < 0.01 and ∗∗p < 0.001 (n = 3).

Article Snippet: For in vivo inhibition of TLR2 in mice liver, C57BL/6 mice were intraperitoneally injected CU-CPT22, a TLR2 antagonist (Selleck.cn).

Techniques: Migration, Activation Assay, Pull Down Assay, Activity Assay, Western Blot, Phospho-proteomics

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet: SAA1/TLR2 axis mediates increased deposition of ECM at injury sites and induces chemokines secretion in activated HSCs (A and B) Sirius red staining of ECM deposition at injury locus in CCl 4 and Cl-induced models. (C) ELISA detection of MCP-1, IL-8, and RANTES secretion from LX-2 cells after treatment of the cells with rhSAA1 for 24 hr. (D) mRNA levels of MCP-1, IL-8, and RANTES. (E) Protein-level detection of MCP-1, IL-8, and RANTES. (Scale bar represents 100 μm). Data represent mean ± SEM ∗p < 0.05, ∗∗p < 0.01 (n = 3).

Article Snippet: For in vivo inhibition of TLR2 in mice liver, C57BL/6 mice were intraperitoneally injected CU-CPT22, a TLR2 antagonist (Selleck.cn).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: SAA1/TLR2 axis directs chemotactic migration of hepatic stellate cells responding to injury

doi: 10.1016/j.isci.2021.102483

Figure Lengend Snippet:

Article Snippet: For in vivo inhibition of TLR2 in mice liver, C57BL/6 mice were intraperitoneally injected CU-CPT22, a TLR2 antagonist (Selleck.cn).

Techniques: Marker, Polymer, Plasmid Preparation, Recombinant, Protease Inhibitor, Saline, Enzyme-linked Immunosorbent Assay, Activation Assay, Silver Staining, Mass Spectrometry, Bicinchoninic Acid Protein Assay, Transfection, Gene Expression, Reporter Assay, Staining, Over Expression, Software, Imaging, Live Cell Imaging, Microscopy

Journal: International Journal of Biological Sciences

Article Title: Itaconate Modulates Neutrophil Homeostasis to Ameliorate Airway Inflammation in Diesel Exhaust Particles-exacerbated Asthma via Inhibiting NETs Formation

doi: 10.7150/ijbs.124927

Figure Lengend Snippet: DEP increases ITA production by inducing metabolic reprogramming in neutrophils. (A) Heatmap of differentially accumulated metabolites between control and HDM/DEP groups, and the change of ITA between control and HDM/DEP groups in the sequencing data. (B) Volcano plot of the differentially expressed genes between the control and HDM/DEP groups. (C) The levels of ITA measured by LC/MS in BALF and lung tissues of HDM/DEP-induced asthma (n = 6-8 mice per group). (D) Real-time qPCR analysis of Acod1 mRNA expression in the lung tissues of HDM/DEP-induced asthma (n = 4 mice per group). (E) Uniform manifold approximation and projection (UMAP) plot showing 13 clusters of cells in the lung tissues. (F) The percentage of the 13 clusters of cells in the lung tissues of con, HDM, and HDM/DEP groups. (G) The expression of Acod1 in different cell clusters. (H) Violin plot of the expression of Acod1 in different cell clusters. (I) Representative immunofluorescence staining of ACOD1 in the lung tissues of DEP-exposed asthmatic mice. Ly6G was used to mark neutrophils, and CD68 to mark macrophages. ACOD1 was co-localized with neutrophils. Scale bar, 20 μm. (J) Real-time qPCR analysis of Acod1 mRNA expression in BMDNs treated with DEP. (K) Representative immunofluorescence staining of ACOD1 in BMDNs treated with DEP. Scale bar, 10 μm. (L) Intracellular and extracellular levels of ITA measured by LC/MS in DEP-stimulated BMDNs. (M) Representative immunofluorescence staining of ACOD1 in cell pellets of BALF from healthy control and patients with severe asthma. Neutrophils were marked with CD16b, and macrophages were marked with CD68. ACOD1 was co-localized with neutrophils. Scale bar, 10 μm. (N) Real-time qPCR analysis of ACOD1 mRNA expression in PBNs stimulated with DEP. (O) Intracellular and extracellular levels of ITA measured by LC/MS in DEP-stimulated PBNs. (P) Integrated analysis of metabolomics and transcriptomics. (Q) Western blot analysis of TLRs and NF-κB signaling pathways in DEP-treated BMDNs. (R) Real-time qPCR analysis of Acod1 mRNA expression in DEP-stimulated BMDNs pre-treated with TLR2 inhibitor (C29), TLR4 inhibitor (TAK-242), and NF-κB inhibitor (BTZ). ** P < 0.01, compared with the control group; ## P < 0.01, ### P < 0.001, compared with the DEP group. (S) Western blot analysis of ACOD1 expression in DEP-stimulated BMDNs pre-treated with TLR2 inhibitor (C29), TLR4 inhibitor (TAK-242), and NF-κB inhibitor (BTZ). Data are presented as means ± SEM. ** P < 0.01, *** P < 0.001. HDM, house dust mite; DEP, diesel exhaust particles; LC/MS, Liquid Chromatography-Mass Spectrometry; BALF, bronchoalveolar lavage fluid; ACOD1, aconitate decarboxylase 1; BMDNs, bone marrow-derived neutrophils; PBNs, peripheral blood neutrophils; TLR, toll-like receptor; BTZ, Bortezomib.

Article Snippet: In some experiments, inhibitors targeting toll-like receptor 2 (TLR2) (C29, 100 μM; MedChemExpress, USA), TLR4 (TAK242, 100 nM; MedChemExpress, USA), the NF-κB signaling pathway (Bortezomib, 50 nM; MedChemExpress, USA), as well as ITA or 4-OI, were administered one hour prior to DEP or LPS treatment.

Techniques: Control, Sequencing, Liquid Chromatography with Mass Spectroscopy, Expressing, Immunofluorescence, Staining, Transcriptomics, Western Blot, Protein-Protein interactions, Liquid Chromatography, Mass Spectrometry, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Virtual Screening Approaches towards the Discovery of Toll-Like Receptor Modulators

doi: 10.3390/ijms17091508

Figure Lengend Snippet: 2D Chemical structure of TLR2 modulators identified by VS techniques and mentioned in this review. The database codes are provided. a MolPort is a supplier of chemicals included in several VS databases ( www.molport.com ).

Article Snippet: ZINC: ZINC398557 [ ] NCI: Plated 2007: 205636 MolPort a : MolPort-001-835-401 TLR2/1 & TLR2/6 inhibitor , C29 [ ] TLR2 TIR domain inhibitor .

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Virtual Screening Approaches towards the Discovery of Toll-Like Receptor Modulators

doi: 10.3390/ijms17091508

Figure Lengend Snippet: Representations of the 3D structures of TLR2/1 ( left ); and TLR2/6 ( right ) complexes with Pam3CSK4 and Pam2CSK4, respectively. The 2D structure of some compounds mentioned in the text are shown.

Article Snippet: ZINC: ZINC398557 [ ] NCI: Plated 2007: 205636 MolPort a : MolPort-001-835-401 TLR2/1 & TLR2/6 inhibitor , C29 [ ] TLR2 TIR domain inhibitor .

Techniques: