Journal: Redox Biology

Article Title: Chemical modification of pro-inflammatory proteins by peroxynitrite increases activation of TLR4 and NF-κB: Implications for the health effects of air pollution and oxidative stress

doi: 10.1016/j.redox.2020.101581

Figure Lengend Snippet: TLR4 activation by native and peroxynitrite-modified proteins. TLR4 activity in HeLa TLR4 dual luciferase reporter cells determined for α-Syn (A), HSP60 (B), HMGB1 (C), and OVA (D) normalized to LPS. Arithmetic mean values and standard deviations of three to four independent experiments performed in triplicates (*p < 0.05, **p < 0.01).

Article Snippet: To inhibit TLR4 or RAGE signaling, cells were pre-incubated with 50 μL of the TLR4 antagonist TAK-242 or the RAGE antagonist FPS-ZM1 for 4 h. TAK-242 (25 mM, Merck Millipore) was provided in dimethyl sulfoxide (DMSO) and further diluted with medium to a final concentration of 0.36 μg mL -1 .

Techniques: Activation Assay, Modification, Activity Assay, Luciferase

Journal: Redox Biology

Article Title: Chemical modification of pro-inflammatory proteins by peroxynitrite increases activation of TLR4 and NF-κB: Implications for the health effects of air pollution and oxidative stress

doi: 10.1016/j.redox.2020.101581

Figure Lengend Snippet: NF-κB activation by native and peroxynitrite-modified proteins. NF-κB activity in THP-1-Lucia™ NF-κB cells determined for α-Syn (A), HSP60 (B), HMGB1 (C), and OVA (D) normalized to LPS. Inhibition experiments with TLR4 antagonist TAK-242 and RAGE antagonist FPS-ZM1. Arithmetic mean values and standard deviations of three to four independent experiments performed in triplicates (*p < 0.05, **p < 0.01).

Article Snippet: To inhibit TLR4 or RAGE signaling, cells were pre-incubated with 50 μL of the TLR4 antagonist TAK-242 or the RAGE antagonist FPS-ZM1 for 4 h. TAK-242 (25 mM, Merck Millipore) was provided in dimethyl sulfoxide (DMSO) and further diluted with medium to a final concentration of 0.36 μg mL -1 .

Techniques: Activation Assay, Modification, Activity Assay, Inhibition

Journal: Experimental & Molecular Medicine

Article Title: IL33-induced neutrophil extracellular traps (NETs) mediate a positive feedback loop for synovial inflammation and NET amplification in rheumatoid arthritis

doi: 10.1038/s12276-024-01351-7

Figure Lengend Snippet: a TLR1 to TLR10 mRNA levels in RA FLSs ( n = 5) treated with 1 ng/μg spon-NETs. b , c Levels of IL-33 and CXCL8 secreted from RA FLSs ( n = 3) preincubated with 20 μM TLR2 antagonist TL2-C29, 1 μM TLR4 antagonist CLI-095 and 3 μM TLR9 antagonist ODN TTAGGG (ODN A151) for 1 h prior to the addition of 1 ng/μL spon-NETs for 24 h. d GSEA of the NF-κB and MAPK (ERK1/2) signaling pathways. e , f Representative western blots showing the levels of phosphorylated ERK1/2, p38, IκBα and p65 in RA neutrophils incubated with 1 ng/μL spon-NETs for 24 h. g , h Levels of IL-33/CXCL8 secreted from RA FLSs ( n = 3) preincubated with the ERK inhibitor PD98059 (1 μM) or the p38 inhibitor SB203580 (10 μM) or the NF-κB inhibitor BAY11-7082 (10 μM) for 1 h prior to the addition of 1 ng/μL spon-NETs for 24 h. i Flowchart of the animal experimental procedures created using BioRender.com. DBA/1J mice received 1.5 mg/mouse type II collagen mAbs via the tail vein on Day 0. On Day 3, 50 µg of LPS was injected via the tail vein to synergize with the mAbs for arthritis development, and intra-articular injections of 3 μM TLR9 antagonist ODN 2088 were administered into the knee joint. The mice were also administered 1 µg of IL-33 intraperitoneally for five consecutive days. j Inflammatory scores of IL-33-induced CAIA mice treated with a TLR9 antagonist ( n = 5) and control mice ( n = 5). k Radiological scores of bone erosion in IL-33-induced CAIA mice treated with a TLR9 antagonist and controls. l citH 3 -DNA levels in the peripheral serum of CAIA model mice treated with a TLR9 antagonist and control mice. m Representative immunofluorescence staining of joint tissue from IL-33-induced CAIA mice treated with a TLR9 antagonist. All the data are presented as the means ± SEMs/SD. Asterisks represent significant differences compared with the reference group, without asterisks. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The inhibitors used included the TLR2 antagonist TL2-C29 (20 μM, Invitrogen), the TLR4 antagonist CLI-095 (1 μM, InvivoGen), the TLR9 antagonist ODN TTAGGG (ODN A151, 3 μM, InvivoGen), the ERK inhibitor PD98059 (1 μM, Selleck), the p38 inhibitor SB203580 (10 μM, Selleck), the NF-κB inhibitor BAY11-7082 (10 μM, Selleck) and DNase I (0.1 mg/mL, Roche).

Techniques: Protein-Protein interactions, Western Blot, Incubation, Injection, Control, Immunofluorescence, Staining

Journal: Experimental & Molecular Medicine

Article Title: IL33-induced neutrophil extracellular traps (NETs) mediate a positive feedback loop for synovial inflammation and NET amplification in rheumatoid arthritis

doi: 10.1038/s12276-024-01351-7

Figure Lengend Snippet: a NET–DNA levels produced from RA neutrophils incubated with various concentrations of IL-33 for 4 h. b Dynamic observation of NET formation in RA neutrophils induced with 100 ng/mL IL-33 using a live cell workstation. c Representative immunofluorescence staining and scanning electron microscopy images of NET formation induced by 100 ng/mL IL-33 in RA neutrophils. d Cellular ROS levels in neutrophils stimulated with 100 ng/mL IL-33, 40 nM PMA, or PBS for 2 h. e NET–DNA levels produced by RA neutrophils pretreated with 10 μM DPI, 10 μM Cl-amidine or 20 μM sivelestat for 1 h prior to stimulation with 100 ng/mL IL-33 or 40 nM PMA for 4 h. f Coimmunoprecipitation assay showing interactions among ST2, MyD88, and TRAF6 in RA neutrophils treated with 100 ng/mL IL-33 for 1 h. g Representative western blots showing ERK1/2, p38, IκBα and p65 phosphorylation levels in RA neutrophils incubated with 100 ng/mL IL-33 for 4 h. h NET–DNA levels induced from RA or HD neutrophils pretreated with 0.2 mg/mL ST2 polyclonal neutralizing antibody for 1 h and then stimulated with 100 ng/mL IL-33 or 40 nM PMA for 4 h. i ST2 mRNA levels in peripheral blood neutrophils from RA patients ( n = 18) and HDs ( n = 18) with or without incubation with 100 ng/mL IL-33 for 12 h. j Flow cytometric analysis of the proportion of CD66b+ST2+ cells among peripheral blood neutrophils from RA patients ( n = 18) and HDs ( n = 18) with or without incubation with 100 ng/mL IL-33 for 12 h. k , l Fold change in the ST2 mRNA level and the proportion of CD66b+ST2+ cells among RA ( n = 18) or HD ( n = 18) neutrophils incubated with 100 ng/mL IL-33 for 12 h. All the data are presented as the means ± SDs/SEMs. Asterisks represent significant differences compared with the reference group, which is without asterisks. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant.

Article Snippet: The inhibitors used included the TLR2 antagonist TL2-C29 (20 μM, Invitrogen), the TLR4 antagonist CLI-095 (1 μM, InvivoGen), the TLR9 antagonist ODN TTAGGG (ODN A151, 3 μM, InvivoGen), the ERK inhibitor PD98059 (1 μM, Selleck), the p38 inhibitor SB203580 (10 μM, Selleck), the NF-κB inhibitor BAY11-7082 (10 μM, Selleck) and DNase I (0.1 mg/mL, Roche).

Techniques: Produced, Incubation, Immunofluorescence, Staining, Electron Microscopy, Co-Immunoprecipitation Assay, Western Blot, Phospho-proteomics

Journal: Experimental & Molecular Medicine

Article Title: IL33-induced neutrophil extracellular traps (NETs) mediate a positive feedback loop for synovial inflammation and NET amplification in rheumatoid arthritis

doi: 10.1038/s12276-024-01351-7

Figure Lengend Snippet: a TLR1 to TLR10 mRNA levels in RA FLSs ( n = 5) treated with 1 ng/μg spon-NETs. b , c Levels of IL-33 and CXCL8 secreted from RA FLSs ( n = 3) preincubated with 20 μM TLR2 antagonist TL2-C29, 1 μM TLR4 antagonist CLI-095 and 3 μM TLR9 antagonist ODN TTAGGG (ODN A151) for 1 h prior to the addition of 1 ng/μL spon-NETs for 24 h. d GSEA of the NF-κB and MAPK (ERK1/2) signaling pathways. e , f Representative western blots showing the levels of phosphorylated ERK1/2, p38, IκBα and p65 in RA neutrophils incubated with 1 ng/μL spon-NETs for 24 h. g , h Levels of IL-33/CXCL8 secreted from RA FLSs ( n = 3) preincubated with the ERK inhibitor PD98059 (1 μM) or the p38 inhibitor SB203580 (10 μM) or the NF-κB inhibitor BAY11-7082 (10 μM) for 1 h prior to the addition of 1 ng/μL spon-NETs for 24 h. i Flowchart of the animal experimental procedures created using BioRender.com. DBA/1J mice received 1.5 mg/mouse type II collagen mAbs via the tail vein on Day 0. On Day 3, 50 µg of LPS was injected via the tail vein to synergize with the mAbs for arthritis development, and intra-articular injections of 3 μM TLR9 antagonist ODN 2088 were administered into the knee joint. The mice were also administered 1 µg of IL-33 intraperitoneally for five consecutive days. j Inflammatory scores of IL-33-induced CAIA mice treated with a TLR9 antagonist ( n = 5) and control mice ( n = 5). k Radiological scores of bone erosion in IL-33-induced CAIA mice treated with a TLR9 antagonist and controls. l citH 3 -DNA levels in the peripheral serum of CAIA model mice treated with a TLR9 antagonist and control mice. m Representative immunofluorescence staining of joint tissue from IL-33-induced CAIA mice treated with a TLR9 antagonist. All the data are presented as the means ± SEMs/SD. Asterisks represent significant differences compared with the reference group, without asterisks. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The inhibitors used included the TLR2 antagonist TL2-C29 (20 μM, Invitrogen), the TLR4 antagonist CLI-095 (1 μM, InvivoGen), the TLR9 antagonist ODN TTAGGG (ODN A151, 3 μM, InvivoGen), the ERK inhibitor PD98059 (1 μM, Selleck), the p38 inhibitor SB203580 (10 μM, Selleck), the NF-κB inhibitor BAY11-7082 (10 μM, Selleck) and DNase I (0.1 mg/mL, Roche).

Techniques: Protein-Protein interactions, Western Blot, Incubation, Injection, Control, Immunofluorescence, Staining

Journal: Theranostics

Article Title: Overexpression of S100A9 in obesity impairs macrophage differentiation via TLR4-NFkB-signaling worsening inflammation and wound healing

doi: 10.7150/thno.67174

Figure Lengend Snippet: S100A9 impairs M2-like macrophage differentiation. A/B) Peritoneal cells were incubated with IL-4 or IL-4+S100A8 homodimer, IL4+S100A9 homodimer or IL-4+S1008/A9 heterodimer. A) Gene expression analysis by quantitative PCR. B) Cytokine levels in the supernatant detected by ELISA. Each symbol represents macrophages from one mouse. Mean is indicated. ANOVA with multiple comparisons *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. C/D) Peritoneal macrophages were incubated with LPS / INFγ or IL-4 to induce a M1 and M2-like macrophage phenotype, respectively. S100A9 was added during M2-like macrophage differentiation. Genome wide expression analysis. Display of the 60 most upregulated gene in (C) M1- versus M2-like macrophages and (D) M2- versus M1-like macrophages. E) Pathway analysis using the KEGG database of differentially expressed proteins and phosphoproteins with a log fold change of > 2 or < 0.5 identified by proteome and phospho-proteome microarray. Macrophages differentiated with IL-4 and IL-4+S100A9 of two different mice were compared. F/G) Detection and quantification of Stat6, p38, p65NFkB, erk1/2, and Akt, phosphorylation in peritoneal macrophages incubated with LPS / INFγ, IL-4 or IL-4 and S100A9 by Western Blot. Rpl-26 served as loading control. Representative Western Blots are shown (F) . Quantification of phosphorylation (G) . Each symbol represents macrophages from one mouse. Mean is indicated. Unpaired t-test or Mann-Whitney test between IL-4 and IL-4+S100A9 treated cells: *P < 0.05, **P < 0.01, ***P < 0.001, ns - not significant. H) Peritoneal macrophages were incubated with IL-4 or IL-4+S100A9 in the presence or absence of TLR4 signaling inhibitor (CLI-095, 1 µM), RAGE antagonistic peptide (ELKVLMEKEL, 10 µM), NFkB inhibitor (BMS-345541, 4 µM), PI3K inhibitor (Apitolisib, 5 µM), or MAPK inhibitor (Cobimitinib, 10 nM). Data represent the mean of ≥ 4 independent experiments. Numbers indicate the mean of AU (RS36-normalized) detected by quantitative PCR and protein detected by ELISA.

Article Snippet: NFkB, MAPK, PI3K, TLR4 and RAGE signaling was inhibited by addition of 4 μM BMS-345541 (Hycultec GmbH, Beutelsbach, Germany), 10 nM Cobitinimib (Selleckchem, Houston, Texas, USA), 5 μM Apitolisib (Selleckchem), 1 μM CLI-095 (Invivogen) and 10 μM RAGE antagonistic peptide (R&D), respectively.

Techniques: Incubation, Gene Expression, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Genome Wide, Expressing, Microarray, Phospho-proteomics, Western Blot, Control, MANN-WHITNEY

Journal: Theranostics

Article Title: Overexpression of S100A9 in obesity impairs macrophage differentiation via TLR4-NFkB-signaling worsening inflammation and wound healing

doi: 10.7150/thno.67174

Figure Lengend Snippet: S100A9 induces IL-1 β secretion in macrophages in the presence of saturated fatty acids. A) Myeloid peritoneal cells from lean chow-fed mice were incubated with 500 µM BSA-complexed PA, stearic acid (SA), or oleic acid (OA), BSA alone or 25 mM glucose (hGlc). IL-1β and TNFα levels within the supernatants were detected by ELISA. B/C) Myeloid peritoneal cells from lean chow-fed mice were incubated with S100A9 in the presence or absence of TLR4 signaling inhibitor (CLI-095, 1 µM), RAGE antagonistic peptide (ELKVLMEKEL, 10 µM), NFkB inhibitor (BMS-345541, 4 µM). B) Relative IL-1β gene expression detected by quantitative PCR; C) IL-1β levels in the supernatant detected by ELISA. D) Myeloid peritoneal cells from lean chow-fed mice were incubated with 500 µM BSA-complexed PA, stearic acid (SA), or oleic acid (OA), or 25 mM glucose followed by stimulation with S100A9. IL-1β and TNFα levels within the supernatants were detected by ELISA. E/F) Myeloid peritoneal cells from lean chow-fed mice were incubated with 500 µM PA-BSA complexes or BSA alone followed by stimulation with S100A9. Inflammasome activation was inhibited by addition of E) 25 µg/ml casapase-1 inhibitor (Ac-YVAD-cmk) or F) 20 mM N-acetyl-L-cysteine (NAC), 10 µM diphenyliodoniumchloride (DPI) or 5 µM Ca074 Me. IL-1β was detected after 24 h by ELISA. Each dot represents one mouse. Mean is indicated. ANOVA with multiple comparisons: *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: NFkB, MAPK, PI3K, TLR4 and RAGE signaling was inhibited by addition of 4 μM BMS-345541 (Hycultec GmbH, Beutelsbach, Germany), 10 nM Cobitinimib (Selleckchem, Houston, Texas, USA), 5 μM Apitolisib (Selleckchem), 1 μM CLI-095 (Invivogen) and 10 μM RAGE antagonistic peptide (R&D), respectively.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Gene Expression, Real-time Polymerase Chain Reaction, Activation Assay

Journal: Theranostics

Article Title: Overexpression of S100A9 in obesity impairs macrophage differentiation via TLR4-NFkB-signaling worsening inflammation and wound healing

doi: 10.7150/thno.67174

Figure Lengend Snippet: S100A9 impairs M2-like macrophage differentiation. A/B) Peritoneal cells were incubated with IL-4 or IL-4+S100A8 homodimer, IL4+S100A9 homodimer or IL-4+S1008/A9 heterodimer. A) Gene expression analysis by quantitative PCR. B) Cytokine levels in the supernatant detected by ELISA. Each symbol represents macrophages from one mouse. Mean is indicated. ANOVA with multiple comparisons *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001. C/D) Peritoneal macrophages were incubated with LPS / INFγ or IL-4 to induce a M1 and M2-like macrophage phenotype, respectively. S100A9 was added during M2-like macrophage differentiation. Genome wide expression analysis. Display of the 60 most upregulated gene in (C) M1- versus M2-like macrophages and (D) M2- versus M1-like macrophages. E) Pathway analysis using the KEGG database of differentially expressed proteins and phosphoproteins with a log fold change of > 2 or < 0.5 identified by proteome and phospho-proteome microarray. Macrophages differentiated with IL-4 and IL-4+S100A9 of two different mice were compared. F/G) Detection and quantification of Stat6, p38, p65NFkB, erk1/2, and Akt, phosphorylation in peritoneal macrophages incubated with LPS / INFγ, IL-4 or IL-4 and S100A9 by Western Blot. Rpl-26 served as loading control. Representative Western Blots are shown (F) . Quantification of phosphorylation (G) . Each symbol represents macrophages from one mouse. Mean is indicated. Unpaired t-test or Mann-Whitney test between IL-4 and IL-4+S100A9 treated cells: *P < 0.05, **P < 0.01, ***P < 0.001, ns - not significant. H) Peritoneal macrophages were incubated with IL-4 or IL-4+S100A9 in the presence or absence of TLR4 signaling inhibitor (CLI-095, 1 µM), RAGE antagonistic peptide (ELKVLMEKEL, 10 µM), NFkB inhibitor (BMS-345541, 4 µM), PI3K inhibitor (Apitolisib, 5 µM), or MAPK inhibitor (Cobimitinib, 10 nM). Data represent the mean of ≥ 4 independent experiments. Numbers indicate the mean of AU (RS36-normalized) detected by quantitative PCR and protein detected by ELISA.

Article Snippet: NFkB, MAPK, PI3K, TLR4 and RAGE signaling was inhibited by addition of 4 μM BMS-345541 (Hycultec GmbH, Beutelsbach, Germany), 10 nM Cobitinimib (Selleckchem, Houston, Texas, USA), 5 μM Apitolisib (Selleckchem), 1 μM CLI-095 (Invivogen) and 10 μM RAGE antagonistic peptide (R&D), respectively.

Techniques: Incubation, Gene Expression, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Genome Wide, Expressing, Microarray, Phospho-proteomics, Western Blot, Control, MANN-WHITNEY

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Contrasting effects of stored allogeneic red blood cells and their supernatants on permeability and inflammatory responses in human pulmonary endothelial cells

doi: 10.1152/ajplung.00025.2019

Figure Lengend Snippet: Analysis of signaling pathways mediating barrier-disruptive effects of red blood cell (RBC) supernatants. A: human pulmonary artery endothelial cells (HPAECs) were pretreated 30 min (first arrow) before RBC supernatant (SUP) addition (second arrow, 1:100 dilution) with vehicle or the following inhibitors: heme inhibitor hemopexin (50 µg/mL and 100 µg/mL) Inset shows transendothelial electrical resistance records in endothelial cell (EC) monolayers treated with hemopexin alone) (left); NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome inhibitor isoliquiritigenin (2 µM), receptor for advanced glycation end-products (RAGE) antagonist FPS-ZM1 (2 µM), and TLR4 inhibitor CLI-095 (5 µM) (middle); and Jak inhibitor I (5 µM), p38 MAPK inhibitor SB203580 (50 µM), and Erk1,2 inhibitor UO126 (5 µM) (right); n = 5. B: effects of the Src inhibitor PP2 (2 µM) and reactive oxygen species (ROS) inhibitor N-acetyl cysteine (NAC; 1 mM) on TER decline caused by fresh RBC supernatants (1:100 dilution); n = 4. C: effects of ROS (NAC, 1 mM), Src (PP2, 2 µM), and RAGE (FPS-ZM1, 2 µM) inhibitors on EC permeability for macromolecules were evaluated using Xpert visualization assay with FITC-avidin as a tracer (green). Nuclear counterstaining with DAPI (blue) demonstrate equal cell densities in control and experimental groups; n = 5; bar = 50 µm. D: NAC and PP2, but not FPS-ZM1, protected EC monolayers against breakdown of cell junctions caused by RBC supernatants, which was monitored by immunofluorescence staining for VE-cadherin; n = 5; bar = 20 µm.

Article Snippet: The following inhibitors were used in cell culture experiments: reactive oxygen species (ROS) inhibitor N -acetyl cysteine and hemopexin used as heme inhibitor (Sigma, St. Louis, MO); NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome inhibitor Isoliquiritigenin and TLR4 inhibitor CLI-095 (InvivoGen, San Diego, CA); Erk1,2 inhibitor UO126 (Promega, Madison, WI); receptor for advanced glycation end-products (RAGE) antagonist FPS-ZM1; Src family inhibitor PP2; JAK inhibitor I (CAS 457081-03-7); and p38MAPK inhibitor SB203580 (EMD Millipore, Billerica, MA).

Techniques: Protein-Protein interactions, Permeability, Avidin-Biotin Assay, Control, Immunofluorescence, Staining