Journal: Neuroscience Bulletin

Article Title: TLR8 in the Trigeminal Ganglion Contributes to the Maintenance of Trigeminal Neuropathic Pain in Mice

doi: 10.1007/s12264-020-00621-4

Figure Lengend Snippet: pIONL causes mechanical allodynia of the ipsilateral facial skin, and increases TLR8 expression in the TG. A Time-course of facial nocifensive behavior score stimulated by a 0.02 g von Frey filament in sham and pIONL mice (** P < 0.01, *** P < 0.001 vs corresponding sham group, two-way RM ANOVA followed by post-hoc Bonferroni test; n = 6 mice/group). B Real-time PCR showing the expression of Tlr8 mRNA in the TG of naïve, sham, and pIONL mice (* P < 0.05, ** P < 0.01, *** P < 0.001 vs sham, Student’s t -test; n = 6 mice/group). C–E Representative TLR8 immunofluorescent images showing the distribution of TLR8 + neurons in the TG of naïve ( C ), sham ( D ), and pIONL 10 days ( E ) mice. F Percentages of TLR8 + cells in the TG of naïve, sham, and pIONL mice (*** P < 0.001 vs sham, one-way ANOVA followed by the Bonferroni test; n = 3 mice/group). G , H Double immunostaining of TLR8 with Tuj1 ( G ) and CD68 ( H ). I Cell-size distribution of TLR8 + neurons and total neurons in the TG 10 days after pIONL. J–M Double-stained immunofluorescent images showing the co-localization of TLR8 with ATF3 ( J ), IB4 ( K ), CGRP ( L ), and NF200 ( M ) in the TG.

Article Snippet: In brief, the sections were blocked by 5% donkey serum in PBS at room temperature for 1 h. Subsequently, the sections were incubated with primary antibodies against TLR8 (rabbit, 1:500, BosterBio), Tuj1 (mouse, 1:2000, R&D Systems), CD68 (rat, 1:1000, AbD Serotec), ATF3 (mouse, 1:500, Santa Cruz), CGRP (mouse, 1:1000, Sigma-Aldrich), and NF200 (mouse, 1:500, Cell Signaling Technology) in a humidified box at 4 °C overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Double Immunostaining, Staining

Journal: Neuroscience Bulletin

Article Title: TLR8 in the Trigeminal Ganglion Contributes to the Maintenance of Trigeminal Neuropathic Pain in Mice

doi: 10.1007/s12264-020-00621-4

Figure Lengend Snippet: Global deletion of Tlr8 or knockdown of Tlr8 in the TG alleviates the mechanical allodynia induced by pIONL. A Nocifensive behavior scores stimulated by a 0.02 g von Frey filament in WT and Tlr8 −/− mice (** P < 0.01, *** P < 0.001 vs WT, two-way RM ANOVA followed by the Bonferroni test). B Nocifensive behavior scores stimulated by a 0.02 g von Frey filament after intra-infraorbital nerve injection of Tlr8 siRNA mixed with RVG 4 days after pIONL (*** P < 0.001 vs NC siRNA, two-way RM ANOVA followed by the Bonferroni test). C RT-PCR of TG tissue showing that Tlr8 siRNA down-regulates Tlr8 mRNA expression (* P < 0.05 vs NC siRNA, Student’s t -test; n = 5 mice/group).

Article Snippet: In brief, the sections were blocked by 5% donkey serum in PBS at room temperature for 1 h. Subsequently, the sections were incubated with primary antibodies against TLR8 (rabbit, 1:500, BosterBio), Tuj1 (mouse, 1:2000, R&D Systems), CD68 (rat, 1:1000, AbD Serotec), ATF3 (mouse, 1:500, Santa Cruz), CGRP (mouse, 1:1000, Sigma-Aldrich), and NF200 (mouse, 1:500, Cell Signaling Technology) in a humidified box at 4 °C overnight.

Techniques: Knockdown, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Neuroscience Bulletin

Article Title: TLR8 in the Trigeminal Ganglion Contributes to the Maintenance of Trigeminal Neuropathic Pain in Mice

doi: 10.1007/s12264-020-00621-4

Figure Lengend Snippet: Expression of pERK, pp38, and pro-inflammatory cytokines induced by pIONL is attenuated in the TG of Tlr8 −/− mice. A , B Phosphorylation of ERK and p38 is increased at 10 days after pIONL in the TG of WT mice (* P < 0.05, ** P < 0.01 vs sham, Student’s t -test; n = 3 mice/group). C , D The phosphorylation of ERK and p38 induced by pIONL is reduced in the TG of Tlr8 −/− mice (* P < 0.05, vs WT, Student’s t -test; n = 3 mice/group). E–G The expression of TNF-α ( E ), IL-1β ( F ), and IL-6 ( G ) is increased by pIONL in the TG of WT mice. The fold-increase of TNF-α ( E ), IL-1β ( F ), and IL-6 ( G ) induced by pIONL is lower in Tlr8 −/− mice than in WT mice (* P < 0.05, ** P < 0.01, *** P < 0.001, pIONL vs sham; # P < 0.05, Tlr8 −/− vs WT-pIONL, Student’s t -test; n = 5–6 mice/group).

Article Snippet: In brief, the sections were blocked by 5% donkey serum in PBS at room temperature for 1 h. Subsequently, the sections were incubated with primary antibodies against TLR8 (rabbit, 1:500, BosterBio), Tuj1 (mouse, 1:2000, R&D Systems), CD68 (rat, 1:1000, AbD Serotec), ATF3 (mouse, 1:500, Santa Cruz), CGRP (mouse, 1:1000, Sigma-Aldrich), and NF200 (mouse, 1:500, Cell Signaling Technology) in a humidified box at 4 °C overnight.

Techniques: Expressing, Phospho-proteomics

Journal: Neuroscience Bulletin

Article Title: TLR8 in the Trigeminal Ganglion Contributes to the Maintenance of Trigeminal Neuropathic Pain in Mice

doi: 10.1007/s12264-020-00621-4

Figure Lengend Snippet: The TLR8 agonist VTX-2337 induces pain hypersensitivity in WT and Tlr7 −/− mice, but not in Tlr8 −/− mice. A Nocifensive behavior scores stimulated by a 0.02 g von Frey filament after VTX-2337 (10, 100, and 500 ng) or PBS injection into the TG in WT mice (* P < 0.05, ** P < 0.01, *** P < 0.001 vs PBS, two-way RM ANOVA followed by Bonferroni test). B Nocifensive behavior scores stimulated by a 0.02 g von Frey filament after VTX-2337 (100 ng) and PBS injection in Tlr8 −/− mice. C Nocifensive behavior scores stimulated by a 0.02 g von Frey filament after VTX-2337 (100 ng) and PBS injection in Tlr7 −/− mice (** P < 0.01, *** P < 0.001 vs PBS, two-way RM ANOVA followed by Bonferroni test).

Article Snippet: In brief, the sections were blocked by 5% donkey serum in PBS at room temperature for 1 h. Subsequently, the sections were incubated with primary antibodies against TLR8 (rabbit, 1:500, BosterBio), Tuj1 (mouse, 1:2000, R&D Systems), CD68 (rat, 1:1000, AbD Serotec), ATF3 (mouse, 1:500, Santa Cruz), CGRP (mouse, 1:1000, Sigma-Aldrich), and NF200 (mouse, 1:500, Cell Signaling Technology) in a humidified box at 4 °C overnight.

Techniques: Injection

Journal: Neuroscience Bulletin

Article Title: TLR8 in the Trigeminal Ganglion Contributes to the Maintenance of Trigeminal Neuropathic Pain in Mice

doi: 10.1007/s12264-020-00621-4

Figure Lengend Snippet: The TLR8 agonist VTX-2337 increases the Ca 2+ concentration in HEK293 cells transfected with TLR8 plasmids and in TG neurons. A , B Effect of VTX-2337 on the F340/380 ratio in HEK293 cells transfected with control plasmids ( A ) or TLR8 plasmids ( B ). C Statistical data showing that VTX-2337 dose-dependently increases the F340/380 fold ratio in HEK293 cells transfected with TLR8 plasmids. D , E Representative Ca 2+ images showing the intracellular Ca 2+ activity in TG neurons at baseline ( D ) and with application of VTX-2337 ( E ) (arrows, neurons responding to VTX-2337). F Representative curves for the F340/380 ratio in TG neurons treated with VTX-2337 (5 μmol/L) in WT mice. G Representative trace of the F340/380 ratio in a TG neuron treated with VTX-2337 (100 nmol/L, 500 nmol/L, and 5 μmol/L) and KCl (50 mmol/L). H Representative trace of the F340/380 ratio in a TG neuron after three treatments with VTX-2337. KCl (50 mmol/L) is used as a positive control. I Percentages of neurons responsive to different concentrations of VTX-2337. J Fold increase of the F340/380 ratio after treatment with different concentrations of VTX-2337. K Representative curves of the F340/380 ratio in TG neurons in Tlr8 −/− mice treated with VTX-2337 (5 μmol/L).

Article Snippet: In brief, the sections were blocked by 5% donkey serum in PBS at room temperature for 1 h. Subsequently, the sections were incubated with primary antibodies against TLR8 (rabbit, 1:500, BosterBio), Tuj1 (mouse, 1:2000, R&D Systems), CD68 (rat, 1:1000, AbD Serotec), ATF3 (mouse, 1:500, Santa Cruz), CGRP (mouse, 1:1000, Sigma-Aldrich), and NF200 (mouse, 1:500, Cell Signaling Technology) in a humidified box at 4 °C overnight.

Techniques: Concentration Assay, Transfection, Control, Activity Assay, Positive Control

Journal: Neuroscience Bulletin

Article Title: TLR8 in the Trigeminal Ganglion Contributes to the Maintenance of Trigeminal Neuropathic Pain in Mice

doi: 10.1007/s12264-020-00621-4

Figure Lengend Snippet: The TLR8 agonist VTX-2337-induced upregulation of pERK, pp38, and pro-inflammatory cytokines is reduced in Tlr8 −/− mice. A , B After intra-TG injection of VTX-2337, the phosphorylation of ERK and p38 is increased in the TG of WT mice (* P < 0.05, ** P < 0.01 vs PBS, Student’s t -test; n = 3 mice/group). C , D The phosphorylation of ERK and p38 in the TG does not increase after intra-TG injection of VTX-2337 in Tlr8 −/− mice ( P > 0.05 vs PBS, Student’s t -test; n = 3 mice/group). E–G The expression of TNF-α ( E ), IL-1β ( F ), and IL-6 ( G ) is increased by intra-TG injection of VTX-2337. The fold-increase of TNF-α ( E ), IL-1β ( F ), and IL-6 ( G ) induced by VTX-2337 is lower in Tlr8 −/− mice than in WT mice (** P < 0.01, *** P < 0.001 vs WT-PBS; # P < 0.05, ## P < 0.01 vs WT-VTX-2337 group, Student’s t -test; n = 5 mice/group).

Article Snippet: In brief, the sections were blocked by 5% donkey serum in PBS at room temperature for 1 h. Subsequently, the sections were incubated with primary antibodies against TLR8 (rabbit, 1:500, BosterBio), Tuj1 (mouse, 1:2000, R&D Systems), CD68 (rat, 1:1000, AbD Serotec), ATF3 (mouse, 1:500, Santa Cruz), CGRP (mouse, 1:1000, Sigma-Aldrich), and NF200 (mouse, 1:500, Cell Signaling Technology) in a humidified box at 4 °C overnight.

Techniques: Injection, Phospho-proteomics, Expressing

Journal: Respiratory Research

Article Title: Oxidative stress augments toll-like receptor 8 mediated neutrophilic responses in healthy subjects

doi: 10.1186/1465-9921-10-50

Figure Lengend Snippet: Detection of toll-like receptor (TLR) 8 in human polymorphonuclear cells (PMNs), and the effects of TLR 7/8 ligand R848 on interleukin(IL)-8 release . (A) TLR8 in PMN was detected by immunocytochemistry. Left panel indicates isotype control. Right panel shows TLR8 immunoreactivity in PMN. (Original magnification: × 400, Scale bars = 10 μm). (B) TLR8 expression was analyzed by flow-cytometry. PMNs were stained by anti-human TLR8 (solid lines) or the isotype control (gray histograms) in the permeabilized (left panel) and unpermeabilized condition (right panel). Left panel indicates both intercellular and cell surface expression of TLR8. Right panel shows cell surface expression alone. (C-F) Effect of R848 on the release of IL-8, and effect of bafilomycin or dexamethasone on the R848-induced IL-8 release from PMN. (C) PMNs were treated with 10 μM R848. The media were harvested at various time points and assayed for IL-8 by ELISA. (D) PMNs were treated for 24 hrs with R837, a ligand of TLR7, or various concentrations of R848, a ligand of TLR 7/8. Media were assayed for IL-8 by ELISA. (E, F) PMNs were treated with 10 μM R848 or vehicle in the presence of various concentrations of bafilomycin, an inhibitor of endosomal acidification (E), or dexamethasone (F). Media were assayed for IL-8 by ELISA. All values are mean values ± SEM of three to four separate experiments. *p < 0.05, **p < 0.01, compared with the values of control; +p < 0.05, ++p < 0.01, compared with the values of the vehicle-pretreated and 10 μM R848-treated group.

Article Snippet: Commercially available reagents were obtained as follows: Mono-Poly Resolving Medium was from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan); fetal calf serum (FCS) and RPMI medium 1640 (RPMI 1640) were from Invitrogen (Carlsbad, California, USA); R848 (resiquimod: 4-amino-2-etoxymethyl-α,α-dimethyl-1 H -imidazo [4,5- c ]quinolin-1-ethanol), bafilomycin and 12-o-tetradecanoylphorbol 13-acetate were from Alexis Biochemicals (San Diego, California, USA); R837 (Imiquimod: 1-isobutyl-1 H -imidazo [4,5- c ]quinolin-4-amine) was from Biomol (Plymouth Meeting, Pennsylvania, USA); N-acethyl- L -cysteine, MG-132, dexamethasone and anti-β-actin antibody were from Sigma (St. Louis, Missouri, USA); anti-TLR8 rabbit polyclonal antibody was from Abgent (San Diego, California, USA); Cellfix solution was from Becton Dickinson (San Jose, California, USA); phycoerythrin (PE)- conjugated anti-TLR8 antibody solution was from Imgenex (San Diego, California, USA); dihydro-rhodamine-123 (DHR-123) was from Cayman Chemical (Ann Arbor, Michigan, USA); human recombinant IL-8 was from Acris antibodies (Hiddenhausen, Germany); anti-human MyD88 antibody, anti-human TRAF6, and anti-human IkBα were from Santa Cruz (San Diego, California, USA); peroxidase-conjugated secondary antibodies were from Rockland Immunochemicals (Gilbertsville, Pennsylvania, USA)

Techniques: Immunocytochemistry, Expressing, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay