Journal: Neuroscience

Article Title: The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

doi: 10.1016/j.neuroscience.2015.09.069

Figure Lengend Snippet: Fig. 1. TLR4 and TLR7 expression within the TG and DRG. Multiple neuronal profiles display intracellular granular IR for TLR4 within the TG (A) and DRG (B), arrows show selected examples of positive neurons. A proportion of non-neuronal cells that were morphologically identified as satellite cells also display a positive stain for TLR4 within both TG and DRG, indicated by asterisks (A, B). Multiple neuronal profiles also display positive IR for TLR7 within the TG (C) and DRG (D), arrows show selected examples. All non-neuronal cells within the tissue are negative for TLR7. Scale bar 50 lm.

Article Snippet: Primary antibodies against TLR4 (1:200, Abcam, Cambridge, UK), TLR7 (1:500, Novus Biologicals, Littleton, CO, USA), LPCAT1 (1:200, ProteinTech, Chicago, IL, USA) or LPCAT2 (1:200, Novus Biologicals, Littleton, CO, USA) were pooled with primary antibodies against either NF200 (1:4000, Sigma–Aldrich, Gillingham, UK), TRPV1 (1:200, Neuromics, Edina, MN, USA) or P2X3 (1:1000, Novus Biologicals, Littleton, CO, USA) and incubated with tissue sections for 24 h, with the exception of TLR4 which required a 48-h incubation, in a humidity chamber at 4 C. After washing with PBS, tissue sections were incubated with a combination of species-specific alexafluor (AF) 488 (10 lg/mL, Life Technologies, Carlsbad, CA, USA), AF555 (10 lg/mL, Life Technologies, Carlsbad, CA, USA) or AF594 (10 lg/mL, Jackson ImmunoResearch, West Grove, PA, USA) conjugated secondary antibodies for 3 h in a dark humidity chamber at room temperature.

Techniques: Expressing, Staining

Journal: Neuroscience

Article Title: The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

doi: 10.1016/j.neuroscience.2015.09.069

Figure Lengend Snippet: Fig. 2. Positive control staining of primary antibodies. Multiple positively stained profiles can be identified for TLR4 (A, B), TLR7 (C, D) and LPCAT2 (E, F) in the spleen and LPCAT1 (G, H) in the lung. Scale bar 15 lm and 50 lm for spleen and lung images, respectively.

Article Snippet: Primary antibodies against TLR4 (1:200, Abcam, Cambridge, UK), TLR7 (1:500, Novus Biologicals, Littleton, CO, USA), LPCAT1 (1:200, ProteinTech, Chicago, IL, USA) or LPCAT2 (1:200, Novus Biologicals, Littleton, CO, USA) were pooled with primary antibodies against either NF200 (1:4000, Sigma–Aldrich, Gillingham, UK), TRPV1 (1:200, Neuromics, Edina, MN, USA) or P2X3 (1:1000, Novus Biologicals, Littleton, CO, USA) and incubated with tissue sections for 24 h, with the exception of TLR4 which required a 48-h incubation, in a humidity chamber at 4 C. After washing with PBS, tissue sections were incubated with a combination of species-specific alexafluor (AF) 488 (10 lg/mL, Life Technologies, Carlsbad, CA, USA), AF555 (10 lg/mL, Life Technologies, Carlsbad, CA, USA) or AF594 (10 lg/mL, Jackson ImmunoResearch, West Grove, PA, USA) conjugated secondary antibodies for 3 h in a dark humidity chamber at room temperature.

Techniques: Positive Control, Staining

Journal: Neuroscience

Article Title: The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

doi: 10.1016/j.neuroscience.2015.09.069

Figure Lengend Snippet: Fig. 3. TLR expression in red blood cells as a negative control. Phase-contrast microscopy was used to identify red blood cells (A). No positive staining was observed in red blood cells for TLR4 (B) or TLR7 (C) antibodies. Scale bar 50 lm.

Article Snippet: Primary antibodies against TLR4 (1:200, Abcam, Cambridge, UK), TLR7 (1:500, Novus Biologicals, Littleton, CO, USA), LPCAT1 (1:200, ProteinTech, Chicago, IL, USA) or LPCAT2 (1:200, Novus Biologicals, Littleton, CO, USA) were pooled with primary antibodies against either NF200 (1:4000, Sigma–Aldrich, Gillingham, UK), TRPV1 (1:200, Neuromics, Edina, MN, USA) or P2X3 (1:1000, Novus Biologicals, Littleton, CO, USA) and incubated with tissue sections for 24 h, with the exception of TLR4 which required a 48-h incubation, in a humidity chamber at 4 C. After washing with PBS, tissue sections were incubated with a combination of species-specific alexafluor (AF) 488 (10 lg/mL, Life Technologies, Carlsbad, CA, USA), AF555 (10 lg/mL, Life Technologies, Carlsbad, CA, USA) or AF594 (10 lg/mL, Jackson ImmunoResearch, West Grove, PA, USA) conjugated secondary antibodies for 3 h in a dark humidity chamber at room temperature.

Techniques: Expressing, Negative Control, Microscopy, Staining

Journal: Neuroscience

Article Title: The expression of Toll-like receptor 4, 7 and co-receptors in neurochemical sub-populations of rat trigeminal ganglion sensory neurons.

doi: 10.1016/j.neuroscience.2015.09.069

Figure Lengend Snippet: Fig. 6. Fluorescence micrograph of rat TG double labeled for TLR7-IR (green, A, D, G) plus either TRPV1- (red, B), P2X3- (red, E) or NF200-IR (red, H). The combined images (C, F, I) show co-localization of TLR7-IR with neurochemical markers. A substantial proportion of TLR7-IR neurons co-express TRPV1-IR, examples of co-expressing neurons are indicated by filled arrows (A, B) and by asterisks in the combined image (C). Some neurons expressing low levels of TRPV1-IR neurons do not display TLR7-IR, examples indicated by open arrows (A, B, C). A substantial proportion of TLR7-IR neurons also co-express P2X3-IR, examples of co-expressing neurons are indicated by filled arrows (D, E) and by asterisks in the combined image (F). Also however, not all P2X3-IR neurons display TLR7-IR, examples indicated by open arrows (D, E, F). There was minimal co- localization between TLR7-IR and NF200-IR; examples of co-expressing neurons are indicated by filled arrows (G, H) and by asterisks in the combined image (I). The vast majority of NF200-IR neurons did not express TLR7-IR, examples indicated by open arrows (G, H, I). A similar pattern of expression and co-expression was observed in the DRG (images not shown). Scale bar 50 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies against TLR4 (1:200, Abcam, Cambridge, UK), TLR7 (1:500, Novus Biologicals, Littleton, CO, USA), LPCAT1 (1:200, ProteinTech, Chicago, IL, USA) or LPCAT2 (1:200, Novus Biologicals, Littleton, CO, USA) were pooled with primary antibodies against either NF200 (1:4000, Sigma–Aldrich, Gillingham, UK), TRPV1 (1:200, Neuromics, Edina, MN, USA) or P2X3 (1:1000, Novus Biologicals, Littleton, CO, USA) and incubated with tissue sections for 24 h, with the exception of TLR4 which required a 48-h incubation, in a humidity chamber at 4 C. After washing with PBS, tissue sections were incubated with a combination of species-specific alexafluor (AF) 488 (10 lg/mL, Life Technologies, Carlsbad, CA, USA), AF555 (10 lg/mL, Life Technologies, Carlsbad, CA, USA) or AF594 (10 lg/mL, Jackson ImmunoResearch, West Grove, PA, USA) conjugated secondary antibodies for 3 h in a dark humidity chamber at room temperature.

Techniques: Fluorescence, Labeling, Expressing

Journal: Archives of Virology

Article Title: Suppression of the toll-like receptor 7-dependent type I interferon production pathway by autophagy resulting from enterovirus 71 and coxsackievirus A16 infections facilitates their replication

doi: 10.1007/s00705-017-3592-x

Figure Lengend Snippet: Autophagy suppresses the TLR7-dependent IFN-I production pathway in EV71 and CA16 infections. A. TLR7-dependent IFN-I mRNA expression examined by qRT-PCR (normalized to GAPDH) in 16HBE cells with different treatments at 0 h, 6 h, 12 h and 24 h. * , P < 0.05; NS, not significant B. TLR7-dependent IFN-I protein expression detected by WB (normalized to GAPDH) in 16HBE cells with different treatments at 0 h, 6 h, 12 h and 24 h. Band intensity values are shown under each band

Article Snippet: After blocking with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature, the PVDF membrane was incubated overnight at 4 °C with the appropriate primary antibody for TLR7 (1:100; Novusbio, USA), MyD88 (1:200; Boster, China), IRF7 (1:1000; Abcam, USA), beclin 1 (1:200; Boster, China), SQSTM1 (1:200; Boster, China), EV71/CA16-VP1 (1:1000; Millipore, USA) or GAPDH (as a loading control, 1:10000; Abmart, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: Archives of Virology

Article Title: Suppression of the toll-like receptor 7-dependent type I interferon production pathway by autophagy resulting from enterovirus 71 and coxsackievirus A16 infections facilitates their replication

doi: 10.1007/s00705-017-3592-x

Figure Lengend Snippet: Autophagy inhibits the TLR7 signaling pathway by disrupting endosome formation. Cells were fixed and permeabilized and then stained with antibodies to TLR7 (green), M6PR (red), or DAPI (blue) (color figure online)

Article Snippet: After blocking with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature, the PVDF membrane was incubated overnight at 4 °C with the appropriate primary antibody for TLR7 (1:100; Novusbio, USA), MyD88 (1:200; Boster, China), IRF7 (1:1000; Abcam, USA), beclin 1 (1:200; Boster, China), SQSTM1 (1:200; Boster, China), EV71/CA16-VP1 (1:1000; Millipore, USA) or GAPDH (as a loading control, 1:10000; Abmart, China).

Techniques: Staining

Journal: PLoS ONE

Article Title: Toll Like Receptor 3 Plays a Critical Role in the Progression and Severity of Acetaminophen-Induced Hepatotoxicity

doi: 10.1371/journal.pone.0065899

Figure Lengend Snippet: ( A ) Quantitative real-time PCR (TaqMan) was performed to measure the transcript levels of TLR3 in whole liver homogenates at 0, 4, 16, 24 and 48 h after the injection of APAP. Data shown indicate mean ± SEM of two independent experiments ( n = 9–10 per time point). ( B ) Confocal immunofluorescent examination of livers revealed TLR3+ cells (red) and the keratin 8+ cells (green) surrounding the areas of necrosis in WT mice at 24 h after APAP injection. Shown are representative sections from 1 mouse of 5 per group. ( C ) The expression of TLR3 was analyzed in primary hepatocytes by western blotting. Each lane represents isolated hepatocytes from independent experiments. ( D ) Expression of TLR3 transcript levels in primary hepatocytes. 1.5×10 5 cells/well were plated in 24 well-plates and stimulated with the indicated stimulus (5 mM of APAP, 20 ng/ml of TNFα, 10 µg/ml of PolyI:C) for 24 h. Bars represents mean±SEM of three independent experiments. *p<0.05 when APAP+TNFα+PolyI:C was compared with APAP+TNFα; ***p<0.0001 when only PolyI:C was compared with medium. Immunofluorescence analysis of livers revealed TLR3+ cells (red) in ( E ) Kupffer cells (F4/80), ( F ) CD34, and ( G ) CD11c in liver sections from WT mice at 24 h after APAP. Magnification: 400×. Shown are representative sections from 1 mouse of 5 per group.

Article Snippet: The following primary antibodies were used: rat anti-mouse keratin 8; rabbit anti-mouse TLR3 ; biotin-labeled anti-mouse F4/80 (Serotec); biotin-labeled anti-mouse CD11c (Biolegend).

Techniques: Real-time Polymerase Chain Reaction, Injection, Expressing, Western Blot, Isolation, Immunofluorescence

Journal: PLoS ONE

Article Title: Toll Like Receptor 3 Plays a Critical Role in the Progression and Severity of Acetaminophen-Induced Hepatotoxicity

doi: 10.1371/journal.pone.0065899

Figure Lengend Snippet: ( A–B ) Representative histological liver samples from WT and tlr3 −/− mice after APAP. ( C ) Lung sections from WT mice at 24 h post APAP. Original magnifications: ( A ) 100× or ( B–C ) 200×. ( A–C ). Data from 4 and 16 h after APAP are representative of 2 independent experiments ( n = 5 mice/group). Data from 24 h after APAP are representative of 5 independent experiments ( n = 5 mice/group), and data from 48 h post APAP is representative of 3 independent experiments (n = 4–9 mice/group). ( D ) Serum ALT and AST levels were measured at various time points after APAP. Values represent mean±SEM obtained from 2–3 independent experiments (n = 4–5 per group) (*p<0.05; **p<0.01). ( E ) Survival of WT and tlr3 −/− mice after 500 mg/kg of APAP. The data are from 2 independent experiments. Each experiment was performed with 10 WT mice and 9–10 tlr3 −/− mice. **p = 0.0037 (Mantel-Cox test). ( F ) Hepatic GSH levels after APAP injection ( n = 3–4 mice/group). Values represent means±SEM of 2 independent experiments. **p<0.01 when tlr3 −/− was compared with WT mice. ( G ) Liver lysates of WT and tlr3 −/− mice were prepared and pooled for Western blotting analysis using anti-Cyp2e1 and β-actin antibodies. ( H ) Concentration of APAP was determined in serum from WT and tlr3 −/− at the indicated time points. Values indicate mean ± SEM of 5–6 individual mice. ns = not significant.

Article Snippet: The following primary antibodies were used: rat anti-mouse keratin 8; rabbit anti-mouse TLR3 ; biotin-labeled anti-mouse F4/80 (Serotec); biotin-labeled anti-mouse CD11c (Biolegend).

Techniques: Injection, Western Blot, Concentration Assay

Journal: PLoS ONE

Article Title: Toll Like Receptor 3 Plays a Critical Role in the Progression and Severity of Acetaminophen-Induced Hepatotoxicity

doi: 10.1371/journal.pone.0065899

Figure Lengend Snippet: Groups of fasted WT mice received APAP (300 mg/kg; i.p. injection), and either rat IgG or anti-TLR3 mAb antibody. Both groups were analyzed at 24 h after APAP injection. ( A ) AST and ALT serum levels from WT mice that received anti-TLR3 mAb 3 h after the APAP injection; enzymes levels from naïve mice were also measured. ( B ) Representative liver sections stained with H&E or ( C ) PAS from WT mice that received either IgG or anti-TLR3 mAb at 3 h after APAP challenge. All tissues were removed at 24 h after APAP challenge. Magnification, 200×. Data in all panels are representative of two independent experiments; n = 5 mice per group.

Article Snippet: The following primary antibodies were used: rat anti-mouse keratin 8; rabbit anti-mouse TLR3 ; biotin-labeled anti-mouse F4/80 (Serotec); biotin-labeled anti-mouse CD11c (Biolegend).

Techniques: Injection, Staining

Journal: PLoS ONE

Article Title: Toll Like Receptor 3 Plays a Critical Role in the Progression and Severity of Acetaminophen-Induced Hepatotoxicity

doi: 10.1371/journal.pone.0065899

Figure Lengend Snippet: Chemokine transcript levels ( A–D ) and protein levels ( E–H ) in WT and tlr3 −/− livers homogenates after APAP. The data represent mean±SEM obtained from 2–3 independent experiments ( n = 5/group). *p<0.05; **p<0.01 and ***p<0.001 when WT were compared with tlr3 −/− mice.

Article Snippet: The following primary antibodies were used: rat anti-mouse keratin 8; rabbit anti-mouse TLR3 ; biotin-labeled anti-mouse F4/80 (Serotec); biotin-labeled anti-mouse CD11c (Biolegend).

Techniques:

Journal: PLoS ONE

Article Title: Toll Like Receptor 3 Plays a Critical Role in the Progression and Severity of Acetaminophen-Induced Hepatotoxicity

doi: 10.1371/journal.pone.0065899

Figure Lengend Snippet: Total liver transcript levels of ( A ) fasl , ( B ) trail , ( C ) tnf and ( D–E ) AP-1 genes in WT and tlr3 −/− mice. ( F ) Total liver TNFα protein levels in WT and tlr3 −/− mice after APAP. Statistically significant differences are indicated (*p<0.05; **p<0.01 and ***p<0.001). Data are presented as the mean ± SEM of 2–3 independent experiments. ( G–H ) Percentage of TNFα+ cells in different liver cell populations isolated from WT and tlr3 −/− mice at 24 h post APAP. Gating is indicated on the left, population statistics are indicated in the bar graph on the right. Rat IgG1, kappa light chain antibody was used as an isotype control for TNFα staining . Representative population of LSECs (CD45 − CD31 + ) is shown. Data is representative of two independent experiments. Data represent mean ± SEM ( n = 3/group in each experiment).

Article Snippet: The following primary antibodies were used: rat anti-mouse keratin 8; rabbit anti-mouse TLR3 ; biotin-labeled anti-mouse F4/80 (Serotec); biotin-labeled anti-mouse CD11c (Biolegend).

Techniques: Isolation, Control, Staining

Journal: PLoS ONE

Article Title: Toll Like Receptor 3 Plays a Critical Role in the Progression and Severity of Acetaminophen-Induced Hepatotoxicity

doi: 10.1371/journal.pone.0065899

Figure Lengend Snippet: ( A ) TNFα protein levels were measured in fasted WT mice that received either control rabbit IgG or anti-TLR3 mAb at 3 h after APAP injection (300 mg/kg; i.p. injection). The data represent mean ± SEM from 2 independent experiments ( n = 5 per group/experiment). *p<0.05; when IgG treated group was compared with anti-TLR3 antibody treatment. ( B ) 2.0×10 4 mouse WT primary hepatocytes were plated in 96-well tissue culture plates and treated with APAP with or without TNFα and/or necrotic RNA. In separate wells, the cells were stimulated only with medium, TNFα or necrotic RNA (no APAP). After 24 h, Presto Blue was added to the cultures and incubated at 37°C, 5% CO 2 . After 1 h, the plates were read for a fluorescence signal that represents viable cells ( n = 12 for each stimulus). ***p<0.001 when medium was compared with APAP; **p<0.01; when APAP alone or APAP+TNFα was compared with APAP+TNFα+necrotic RNA. #p<0.05 when necrotic RNA was compared with medium; ####p<0.0001 when TNFα was compared with medium.

Article Snippet: The following primary antibodies were used: rat anti-mouse keratin 8; rabbit anti-mouse TLR3 ; biotin-labeled anti-mouse F4/80 (Serotec); biotin-labeled anti-mouse CD11c (Biolegend).

Techniques: Control, Injection, Incubation, Fluorescence

Journal: PLoS ONE

Article Title: Toll Like Receptor 3 Plays a Critical Role in the Progression and Severity of Acetaminophen-Induced Hepatotoxicity

doi: 10.1371/journal.pone.0065899

Figure Lengend Snippet: ( A ) Phosphorylation of JNK in liver samples was measured after APAP. Liver lysates of WT and tlr3 −/− mice or ( B ) NmuLi were analyzed and the immunoblots shown are representative of three independent experiments while the immunoblots from 30′ time point are representative of four independent experiments. ( C ) The data in ( B ) were quantified by densitometry analysis using Image J Software. ( D ) Confocal analysis indicating the expression of p-JNK (red) and Keratin 8 + (green) in hepatocytes from WT mice that did not receive APAP (control), and that received either rabbit IgG or anti-TLR3 polyclonal antibody 3 h after APAP injection. DAPI stained the nuclei (blue). Tissues were collected 4 h post APAP injection. Representative images at 400 x are shown ( n = 5 per group).

Article Snippet: The following primary antibodies were used: rat anti-mouse keratin 8; rabbit anti-mouse TLR3 ; biotin-labeled anti-mouse F4/80 (Serotec); biotin-labeled anti-mouse CD11c (Biolegend).

Techniques: Phospho-proteomics, Western Blot, Software, Expressing, Control, Injection, Staining

Journal: eLife

Article Title: A zebrafish and mouse model for selective pruritus via direct activation of TRPA1

doi: 10.7554/eLife.32036

Figure Lengend Snippet: ( A, B ) In situ hybridization in 3dpf WT larvae probing for tlr7 and trpa1b mRNA respectively. ( C, D ) Larval locomotor assay of 5dpf tlr7 +/+ and tlr7 -/- ( C ) or trpa1b +/+ and trpa1b -/- ( D ) larvae. ( E, F ) Adult lip-rubbing behavioral assay of tlr7 +/+ /tlr7 -/- ( E ) and trpa1b +/+ /trpa1b -/- ( F ) fish. ( C ), ( D ), ( E ), ( F ), 100 μM IMQ used. ***p<0.001, **p<0.01, Student’s t -test. Bars represent mean ± s.e.m. ( G, H ) Representative calcium imaging traces of 3dpf trpa1b +/+ ( G ) and trpa1b -/- ( H ) larvae in a transgenic elavl3 HuC:GCaMP5 background exposed to 100 μM IMQ, 50 μM AITC, and 1 mM 2-APB. B = blood, TG = trigeminal ganglion.

Article Snippet: Primary antibodies against TLR7 (rabbit anti-TLR7, Boster, Pleasanton, CA, 1:250) and GFP (chick anti-GFP, 1:1000, Invitrogen) were made in PBST with 10% goat serum and applied to the coverslips, which were incubated overnight at 4°C.

Techniques: In Situ Hybridization, Behavioral Assay, Imaging, Transgenic Assay

Journal: eLife

Article Title: A zebrafish and mouse model for selective pruritus via direct activation of TRPA1

doi: 10.7554/eLife.32036

Figure Lengend Snippet: ( A ) The 7 bp deletion in the trpa1b coding sequence generated a premature stop codon at 1412 bp. ( B ) Amino acid sequences of the WT and mutant Trpa1b proteins. ( C ) In situ hybridization probing for trpa1b mRNA in WT 3dpf larval zebrafish, demonstrating expression in RB neurons. ( D ) Schematic of the Trpa1b protein structure, demonstrating that a truncated protein (in the unlikely event that it was translated) would lack a critical cysteine residue required for agonist binding . ( E ) Trpa1b -/- nonsense mutants locomoted more in response to increasing temperatures at levels equivalent to their WT/heterozygous siblings. ( F ) Normal AITC behavioral responses (increased locomotion) are abolished in trpa1b -/- nonsense mutants. ( G ) The 1 bp deletion in the tlr7 coding sequence generated a premature stop codon at bp 665. ( H ) Amino acid sequences of WT and mutant Tlr7 proteins. ( I ) Schematic of the Tlr7 protein, demonstrating that a truncated protein (in the unlikely event of translation) would lack critical functional domains. ( J ) In situ hybridization probing for tlr7 mRNA in WT 3dpf larval zebrafish. No tlr7 expression was observed in RB somatosensory neurons. Premature stops are denoted with red highlighting. EC = extracellular, IC = intracellular, TM = transmembrane domain, LRR = leucine rich repeat, TIR = Toll Interleukin-1 Resistance domain. C and N denote c- and n-termini. ( E ), Student’s t -test.

Article Snippet: Primary antibodies against TLR7 (rabbit anti-TLR7, Boster, Pleasanton, CA, 1:250) and GFP (chick anti-GFP, 1:1000, Invitrogen) were made in PBST with 10% goat serum and applied to the coverslips, which were incubated overnight at 4°C.

Techniques: Sequencing, Generated, Mutagenesis, In Situ Hybridization, Expressing, Residue, Binding Assay, Functional Assay

Journal: eLife

Article Title: A zebrafish and mouse model for selective pruritus via direct activation of TRPA1

doi: 10.7554/eLife.32036

Figure Lengend Snippet: ( A–C ) Calcium imaging of HEK cells transfected with zebrafish ( A ), mouse ( B ), and human ( C ) Trpa1 or Trpa1 + Tlr7 . ( D, E, F ) Calcium imaging dose response curves of HEK cells transfected with zebrafish ( D ), mouse ( E ), and human ( F ) Trpa1 , exposed to IMQ or AITC. ( A–F ) Numbers represent total cell counts per condition. ( G ) Patch clamp of HEK cell transfected with zebrafish trpa1b exposed to 100 μM IMQ. ( H ) Patch clamp dose response curve for HEK cells transfected with zebrafish trpa1b , mouse Trpa1 , or mouse Trpa1 + mouse Tlr7 . ( I ) Current density values of HEK cells exposed transfected with zebrafish trpa1b or mouse Trpa1 and exposed to 100 μM AITC or 100 μM IMQ. ( G–I ) n = 5 cells per condition. **p<0.01, Student’s t -test. Bars represent mean ± s.e.m.

Article Snippet: Primary antibodies against TLR7 (rabbit anti-TLR7, Boster, Pleasanton, CA, 1:250) and GFP (chick anti-GFP, 1:1000, Invitrogen) were made in PBST with 10% goat serum and applied to the coverslips, which were incubated overnight at 4°C.

Techniques: Imaging, Transfection, Patch Clamp

Journal: eLife

Article Title: A zebrafish and mouse model for selective pruritus via direct activation of TRPA1

doi: 10.7554/eLife.32036

Figure Lengend Snippet: ( A–D ) Average traces from calcium imaging experiments with HEK cells transiently transfected with zebrafish trpa1b ( n = 85) ( A ), zebrafish trpa1b + zebrafish tlr7 ( n = 80) ( B ), mouse Trpa1 ( n = 134) ( C ), or mouse Trpa1 + mouse Tlr7 ( n = 89) ( D ). n = 22, 23, 10, and 19 untransfected control HEK cells per experiment, respectively. No gross differences were observed between the two conditions for each species. ( E ) Quantification of peak fluorescence intensity achieved during stimulation with 100 μM IMQ across all HEK cell transfection conditions. In all species examined, no significant difference was observed between cells transfected with only Trpa1 and cells transfected with Trpa1 plus the corresponding Tlr7 . ( F–H ), Immunohistochemistry performed on HEK cells transfected with pIRES-eGFP only ( F ), mouse Tlr7 ( G ), or human TLR7 ( H ). As shown, TLR7 labeling (red) was only observed HEK cells transfected with Tlr7 constructs. ( I–J ), I/V curves from voltage clamp experiments using cells transfected with mTRPA1 ( I ) and mouse Trpa1 + mouse Tlr7 ( J ). As shown in ( I ), 100 μM IMQ elicited greater current influx in transfected cells than under basal conditions, an effect that disappeared during washout. Cells transfected with both mouse Trpa1 and mouse Tlr7 also showed significant current flux during IMQ stimulus, but this was not different from cells transfected only with mTRPA1. n = 5 cells per condition. ( K ) a table of EC50 values for both IMQ and AITC stimuli in cells transfected with zebrafish trpa1b , mouse Trpa1 , and human TRPA1 in . As shown, the EC50 values for IMQ are much greater than those of AITC for all species of TRPA1. ΔF/F (fluorescence intensity change) is expressed as a normalized 340 nm/380 nm intensity ratio. (*p<0.05, **p<0.01, ***p<0.001, Student’s t -test. Bars are expressed as means ± s.e.m.

Article Snippet: Primary antibodies against TLR7 (rabbit anti-TLR7, Boster, Pleasanton, CA, 1:250) and GFP (chick anti-GFP, 1:1000, Invitrogen) were made in PBST with 10% goat serum and applied to the coverslips, which were incubated overnight at 4°C.

Techniques: Imaging, Transfection, Control, Fluorescence, Immunohistochemistry, Labeling, Construct

Journal: eLife

Article Title: A zebrafish and mouse model for selective pruritus via direct activation of TRPA1

doi: 10.7554/eLife.32036

Figure Lengend Snippet: ( A ) A comparison of average peak fluorescence values obtained from Trpa1 or Trpa1 + Tlr7 transfected HEK cells in calcium imaging experiments following stimulation by 100 μM IMQ or 100 μM loxoribine. ( B, C ) Average traces from calcium experiments in which HEK cells were transfected with zebrafish trpa1b + zebrafish tlr7 ( n = 102) ( B ) or mouse Trpa1 + mouse Tlr7 ( n = 45) ( C ) and treated with 100 μM loxoribine and 100 μM AITC. 15 and 21 untransfected control cells were present in each respective experiment. As shown, loxoribine did not elicit any calcium flux. ( D ) I/V curve from voltage clamp experiments in which HEK cells were transfected with mouse Trpa1 + mouse Tlr7 and stimulated with both loxoribine (100 μM) and AITC (100 μM). While AITC elicited remarkable current influx, current change associated with application of loxoribine did not change current flow above baseline levels. ( E ) A dual luciferase assay to verify the functionality of transfected TLR7 and loxoribine. Cells transfected with only the two luciferase constructs and pIRES-eGFP did not demonstrate any NF-kB induction following stimulation with 200 μM loxoribine, as expected. Notable NF-kB induction was observed in cells transfected with the two luciferase constructs and either mouse Tlr7 or human TLR7 following stimulation with loxoribine. Intriguingly, cells transfected with the zebrafish tlr7 did not exhibit any significant NF-kB induction following application of loxoribine. ( F ) A dual luciferase assay in which both pIRES-eGFP-transfected control HEK cells and zebrafish tlr7 -transfected cells were treated with TLR7 agonists loxoribine (500 μM and 1 mM) and IMQ (100 μM and 500 μM). There was no difference in NF-kB induction between pIRES-eGFP-only and zebrafish tlr7- transfected cells, potentially indicating that zebrafish Tlr7 is unresponsive to typical mammalian TLR7 agonists. ΔF/F (fluorescence intensity change) is expressed as a normalized 340 nm/380 nm intensity ratio. Luminosity values are expressed as the firefly/renilla ratio of relative light units in counts per second (CPS) following background subtraction. *p<0.05, **p<0.01, ***p<0.001, Student’s t -test ( A, E ) or one-way ANOVA ( F ). Bars are expressed as means ± s.e.m.

Article Snippet: Primary antibodies against TLR7 (rabbit anti-TLR7, Boster, Pleasanton, CA, 1:250) and GFP (chick anti-GFP, 1:1000, Invitrogen) were made in PBST with 10% goat serum and applied to the coverslips, which were incubated overnight at 4°C.

Techniques: Comparison, Fluorescence, Transfection, Imaging, Control, Luciferase, Construct

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: EGF Receptor-dependent TLR7 Signaling in Macrophages Promotes Glomerular Injury in Crescentic Glomerulonephritis

doi: 10.1016/j.labinv.2023.100190

Figure Lengend Snippet: (A) Wild type (WT) or TLR7−/− mice were primed with 0.5 mg of normal rabbit IgG emulsified with Incomplete Freund’s Adjuvant (IFA) on Day −5, followed by intravenous injection with 50 ul of nephrotoxic serum on Day 0. Spot urine was collected on Day 7 to measure urine protein creatinine ratio. Kidneys were harvested on Day 7 to evaluate fibrinoid necrosis (B) and crescent formation (C). (D) A representative image of crescent formation in glomerulus from a WT mouse. Periodic acid-methenamine silver staining. (E) A representative image of fibrinoid necrosis (glomerular basement membrane rupture, fibrin deposits, and karyorrhexis) in glomerulus from a WT mouse. Periodic acid-methenamine silver staining. (F) A representative image of unremarkable glomerulus from a TLR7−/− mouse. Periodic acid-methenamine silver staining. (G) Neutrophils were stained with esterase on the kidney tissue sections from WT mice and TLR7−/− mice 2 hrs after NTS challenge. The number of esterase positive cells were counted in all glomeruli. (H) A representative image of esterase positive cells in glomeruli of WT mice and TLR7−/− mice. (I) Macrophages were stained with anti-Mac-2 Ab on the kidney tissue sections from WT mice and TLR7−/− mice 7 days after NTS challenge. The number of Mac-2 positive cells were counted in all glomeruli. (J) A representative image of Mac-2 positive cells in glomeruli of WT mice and TLR7−/− mice. All yellow bars: 25 μm; *: P<0.05; **: P<0.005.

Article Snippet: Immunohistochemistry and esterase staining After antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) or 0.76 g/L pronase in TBS for 30 min, intrinsic peroxidases were inactivated by incubation in 3% hydrogen peroxidase for 10 min. To reduce non-specific background staining, sections were treated with 1% bovine serum albumin (BSA) for 30 min and Streptavidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA) for 15 min each and then incubated for 24 h at 4°C with biotin anti-mouse/human Mac-2 (BioLegend, San Diego, CA) or biotin anti-TLR7 (G-BIOSCIENCES, St. Louis, MO), followed by the incubation with a horseradish peroxidase streptavidin (Vector Laboratories, Burlingame, CA) for 60 min at room temperature.

Techniques: Adjuvant, Injection, Silver Staining, Membrane, Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: EGF Receptor-dependent TLR7 Signaling in Macrophages Promotes Glomerular Injury in Crescentic Glomerulonephritis

doi: 10.1016/j.labinv.2023.100190

Figure Lengend Snippet: (A) Autologous murine anti-rabbit antibody levels were determined by ELISA from serum samples from WT (n=8) or TLR7−/− (n=8) mice on Day 7 of NTN. Titers in serial dilutions of serum are expressed in arbitrary units (OD 450 nm). (B) Splenocytes were isolated from WT or TLR7−/− mice after 14 days immunization of rabbit IgG. Cells were incubated with or without rabbit IgG for 24 h. Supernatants were collected to measure IL-6 and IFN-γ by ELISA. (C) Representative immunofluorescence images of glomerular mouse IgG (upper panels) and rabbit IgG (lower panels) depositions in WT (left panels) or TLR7−/− (right panels) mice on day 7 of NTN. Scale bars = 25 μm. (D) Semi-quantitative analysis of mouse IgG (upper panel) and rabbit IgG (lower panel) depositions in glomeruli of WT or TLR7−/− mice on day 7 of NTN. Representative Electron microscopy images of glomerular capillary loops (E) and mesangial areas (F) from WT mice and TLR7−/− mice with abundant subendothelial electron dense immune-complex deposits and swollen endothelial cells, and numerous electron dense deposits in mesangial areas. Yellow asterisks indicate deposits. [uranyl acetate and lead citrate stain, scale bars: 1 μm].

Article Snippet: Immunohistochemistry and esterase staining After antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) or 0.76 g/L pronase in TBS for 30 min, intrinsic peroxidases were inactivated by incubation in 3% hydrogen peroxidase for 10 min. To reduce non-specific background staining, sections were treated with 1% bovine serum albumin (BSA) for 30 min and Streptavidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA) for 15 min each and then incubated for 24 h at 4°C with biotin anti-mouse/human Mac-2 (BioLegend, San Diego, CA) or biotin anti-TLR7 (G-BIOSCIENCES, St. Louis, MO), followed by the incubation with a horseradish peroxidase streptavidin (Vector Laboratories, Burlingame, CA) for 60 min at room temperature.

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Incubation, Immunofluorescence, Electron Microscopy, Staining

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: EGF Receptor-dependent TLR7 Signaling in Macrophages Promotes Glomerular Injury in Crescentic Glomerulonephritis

doi: 10.1016/j.labinv.2023.100190

Figure Lengend Snippet: (A) TLR7 expression level was examined by Western Blot in various mouse cells. RAW: RAW 264.7 cells (a macrophage cell line), BMM: bone marrow-derived macrophages, MES: primary mesangial cells, and POD: temperature-sensitive SV40 T antigen-expressing podocytes. (B) TLR7 expression level was tested by Western Blot in human cells. THP-1: a macrophage cell line, MES: primary mesangial cells, and POD: temperature-sensitive SV40 T antigen-expressing podocytes. (C) Mouse podocytes were incubated with LPS (0.01 μg/ml), R848 (10 μg/ml), or left untreated for 24 hrs. The supernatants were collected to measure IL-6 by ELISA. **: P<0.005. (D) Neutrophils (Neu) and peritoneal macrophages (pMΦ) were collected from WT mice or TLR7−/− mice. TLR7 expression level was tested by Western Blot. (E) Expression of TLR7 in RAW 264.7 cells and isolated glomeruli from WT or TLR7−/− mice were analyzed by Western blot. (F) Immunohistochemistry of TLR7 in glomerular infiltrates from a WT NTN mouse.

Article Snippet: Immunohistochemistry and esterase staining After antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) or 0.76 g/L pronase in TBS for 30 min, intrinsic peroxidases were inactivated by incubation in 3% hydrogen peroxidase for 10 min. To reduce non-specific background staining, sections were treated with 1% bovine serum albumin (BSA) for 30 min and Streptavidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA) for 15 min each and then incubated for 24 h at 4°C with biotin anti-mouse/human Mac-2 (BioLegend, San Diego, CA) or biotin anti-TLR7 (G-BIOSCIENCES, St. Louis, MO), followed by the incubation with a horseradish peroxidase streptavidin (Vector Laboratories, Burlingame, CA) for 60 min at room temperature.

Techniques: Expressing, Western Blot, Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Isolation, Immunohistochemistry

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: EGF Receptor-dependent TLR7 Signaling in Macrophages Promotes Glomerular Injury in Crescentic Glomerulonephritis

doi: 10.1016/j.labinv.2023.100190

Figure Lengend Snippet: (A) RAW 264.7 cells were stimulated with R848 (0.1 μg/ml) for 60 min with or without Gefitinib (20 μM) preincubation for 60 min, or left untreated. p65 was stained to identify p65 localization (scale bars: 20 μm), and the nuclear positive cells for p65 were counted. (B) RAW 264.7 cells were incubated with IFN-γ (1000 U/ml), a TLR3 ligand (poly (I:C), 10 μg/ml), a TLR7 ligand (R848, 0.1 μg/ml) or a TLR9 ligand (CpGB, 0.1μM) in the presence or absence of an EGFR inhibitor (Gefitinib, 20 μM) for 24 hrs. IL-6 in the culture media was measured by ELISA. (C) Bone marrow-derived macrophages were incubated with poly(I:C), R848 or CpGB in the presence or absence of Gefitinib for 24 hrs. IL-6 in the culture media was measured by ELISA. (D) RAW 264.7 cells were stimulated by R848 (0.1 μg/mL) for 1 hour with or without pre-treatment of Gefitinib (10 or 20 μM). Expressions of phospho-IκBα, total IκBα, and actin were tested by Western blot. (E) RAW 264.7 cells were stimulated by R848 (0.1 μg/mL) for 8 hours with or without pre-treatment of Gefitinib (10 or 20 μM). Expressions of p56 and Actin were tested by Western blot.

Article Snippet: Immunohistochemistry and esterase staining After antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) or 0.76 g/L pronase in TBS for 30 min, intrinsic peroxidases were inactivated by incubation in 3% hydrogen peroxidase for 10 min. To reduce non-specific background staining, sections were treated with 1% bovine serum albumin (BSA) for 30 min and Streptavidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA) for 15 min each and then incubated for 24 h at 4°C with biotin anti-mouse/human Mac-2 (BioLegend, San Diego, CA) or biotin anti-TLR7 (G-BIOSCIENCES, St. Louis, MO), followed by the incubation with a horseradish peroxidase streptavidin (Vector Laboratories, Burlingame, CA) for 60 min at room temperature.

Techniques: Staining, Incubation, Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: EGF Receptor-dependent TLR7 Signaling in Macrophages Promotes Glomerular Injury in Crescentic Glomerulonephritis

doi: 10.1016/j.labinv.2023.100190

Figure Lengend Snippet: Nephrotoxic serum nephritis was induced in wild type mice, TLR7−/− mice, or mice lacking EGFR in macrophages. Twelve hours before injection of nephrotoxic serum, some mice were treated with vehicle or 2.4 g/day of gefitinib by oral gavage (Day −1 to 7). Spot urine was collected on Day 7 to measure urine protein creatinine ratio (A). Kidneys were harvested on Day 7, and glomeruli with fibrinoid necrosis (B) and crescents (C) and glomerular Mac-2 positive MΦs (D) were evaluated. n.s.: no significant difference; (A) *: P<0.01 for TLR7+/+ without gefitinib vs. EKOMΦ, and P<0.05 for TLR7−/− without gefitinib vs. EKOMΦ. **: P = 0.0044; (B) and (D) ****: P < 0.0001; (C) **: P = 0.0010 (E) Glomerular macrophages were purified from several different strains and conditions as indicated, on Day 7. P56 induction was measured by Western Blot (n = 5, each) (F) GFR was measured in the same groups of mice on Day 7. **: P = 0.0078, TLR7+/+ vs. TLR7-/−. (G) Role of TLR7-EGFR axis in macrophages in crescentic GN. (1) Nephrotoxic serum induces injuries in glomerular resident cells and inflammatory cells, releasing endogenous TLR7 ligands. (2) TLR7 ligands activate TLR7 in macrophages, which requires EGFR kinase activity to phosphorylates TLR7 tyrosine residues. Upon EGFR-mediated TLR7 activation, macrophages exhibit cell migration enhancement (3) and inflammatory cytokine production (4), leading to crescent formation, a malignant histological lesion. (5) EGFR inhibition efficiently blocks TLR7 signaling, and subsequent glomerular damages.

Article Snippet: Immunohistochemistry and esterase staining After antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) or 0.76 g/L pronase in TBS for 30 min, intrinsic peroxidases were inactivated by incubation in 3% hydrogen peroxidase for 10 min. To reduce non-specific background staining, sections were treated with 1% bovine serum albumin (BSA) for 30 min and Streptavidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA) for 15 min each and then incubated for 24 h at 4°C with biotin anti-mouse/human Mac-2 (BioLegend, San Diego, CA) or biotin anti-TLR7 (G-BIOSCIENCES, St. Louis, MO), followed by the incubation with a horseradish peroxidase streptavidin (Vector Laboratories, Burlingame, CA) for 60 min at room temperature.

Techniques: Injection, Purification, Western Blot, Activity Assay, Activation Assay, Migration, Inhibition

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: EGF Receptor-dependent TLR7 Signaling in Macrophages Promotes Glomerular Injury in Crescentic Glomerulonephritis

doi: 10.1016/j.labinv.2023.100190

Figure Lengend Snippet: (A) Raw 264.7 cells were incubated with R848 at 1.0 μg/ml for indicated time, and cell lysates were immunoprecipitated with anti-EGFR Ab. TLR7-EGFR interaction was detected with anti-TLR7 Ab Western Blot. The expression of EGFR or actin in original cell lysates was analyzed by Western Blot. (B) Raw 264.7 cells were incubated with R848 at 1.0 μg/ml for 30 min. The lysates were immunoprecipitated with anti-phosphotyrosine Ab. Tyrosine phosphorylation of TLR7 and actin (loading control) were detected by anti-TLR7 Ab and anti-actin Ab, respectively, by Western Blot. (C) Raw 264.7 cells were incubated with R848 ± gefitinib (20 μM), and cell lysates were immunoprecipitated with anti-phosphotyrosine Ab, followed by Western Blot with anti-TLR7 Ab to detect tyrosine phosphorylation of TLR7. Actin was used for loading control. (D) Bone marrow-derived macrophages were used for the same experiment as in (C).

Article Snippet: Immunohistochemistry and esterase staining After antigen retrieval by heating in 0.01 M citrate buffer (pH 6.0) or 0.76 g/L pronase in TBS for 30 min, intrinsic peroxidases were inactivated by incubation in 3% hydrogen peroxidase for 10 min. To reduce non-specific background staining, sections were treated with 1% bovine serum albumin (BSA) for 30 min and Streptavidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA) for 15 min each and then incubated for 24 h at 4°C with biotin anti-mouse/human Mac-2 (BioLegend, San Diego, CA) or biotin anti-TLR7 (G-BIOSCIENCES, St. Louis, MO), followed by the incubation with a horseradish peroxidase streptavidin (Vector Laboratories, Burlingame, CA) for 60 min at room temperature.

Techniques: Incubation, Immunoprecipitation, Western Blot, Expressing, Derivative Assay