Journal: The FASEB Journal

Article Title: SR‐717, a Non‐Nucleotide STING Agonist, Displayed Anti‐Radiation Activity in a IL‐6 Dependent Manner

doi: 10.1096/fj.202403127R

Figure Lengend Snippet: SR‐717 plays a role in intestinal radiation protection through TLR2 signaling pathway. (A) The KEGG pathway top 10 (Total) enrichment in the intestines of mice treated in the presence or absence of SR‐717. (B) GSEA of pattern recognition receptor activity. (C) GSEA of Tolls signaling pathway. (D) Heatmap of Toll‐like receptor signaling pathway genes. (E) RNA obtained from mice intestinal was used to perform the qRT‐PCR ( n = 3). (F) Western blot analysis of activation of the TLR2 signaling pathways in MODE‐K cells stimulated with SR‐717 ( n = 3). (G) TLR2 KO mice were treated with SR‐717 or saline before 9.5 Gy TBI. Pathological images and statistical analysis of the intestine in mice ( n = 3). (H) Organoid extracted from TLR2 KO mice, were then exposed to SR‐717 or PBS. And the relative area and budding condition of intestinal organoids. Data are represented as mean ± SEM. ns, non‐significance.

Article Snippet: Wild‐type (WT) littermates and TLR2 KO mice, 6–8 weeks old, were purchased from Cyagen (Jiangsu, China).

Techniques: Activity Assay, Quantitative RT-PCR, Western Blot, Activation Assay, Protein-Protein interactions, Saline

Journal: Nature Communications

Article Title: Microglia clear neuron-released α-synuclein via selective autophagy and prevent neurodegeneration

doi: 10.1038/s41467-020-15119-w

Figure Lengend Snippet: a WT microglia were treated with 250 nM h α-Syn protein for the indicated time and assayed for W.B using antibodies as indicated. The levels of proteins were quantified in the right panel ( n = 6). p -values were calculated by one-way ANOVA with Newman–Keuls post hoc test. F(4,24) = 9.196, p = 0.0001 for p-NF-κB, F(4,24) = 4.530, p = 0.0072 for IkB, F(4,24) = 0.8644, p = 0.4994 for p-ERK1/2, F(4,24) = 1.369, p = 0.2741 for p-JNK, F(4,24) = 5.372, p = 0.0031 for p-p38. b , c Cells were pretreated with ML-120B, an IKK-2 inhibitor, or SB203580 and SB202190, two different p38 inhibitors, for indicated concentrations before 250 nM h α-Syn protein treatment, and assayed for W.B ( b , n = 3–5, F(3,12) = 3.750, p = 0.0413) or immunostaining ( c , n = 33, F(4,160) = 56.21, p = 1.53E-29). The levels of proteins were quantified in the right panel. The number of h α-Syn/ubiquitin-positive puncta was counted and quantified ( c ). p -values were calculated by one-way ANOVA with Newman–Keuls post hoc test. d – f Microglia obtained from WT mice and Tlr4 -KO mice were treated with h α-Syn protein for 3 h with indicated concentration without ( d , n = 6) or with ( e ( n = 6), f ( n = 4)) TLR2-blocking antibody (T2.5), and assayed for RT-qPCR using primers for Il-1b (left panel, F(3,40) = 20.70, p = 2.95E-08 for hα-Syn-genotype interaction; F(3,40) = 69.43, p = 6.55E-16 for hα-Syn; F(1,40) = 69.57, p = 2.74E-10 for genotype) and Tnf (right panel, F(3,40) = 10.12, p = 4.32E-05 for hα-Syn-genotype interaction; F(3,40) = 21.73, p = 1.64E-08 for hα-Syn; F(1,40) = 34.36, p = 7.39E-07 for genotype). p -values were calculated by two-way ANOVA with Bonferroni post hoc test (wt vs ko in d ), one-way ANOVA with Newman–Keuls post hoc test (non-treated vs. treated in d , e ; F(3,20) = 22.11, p = 1.46E-06 for Il-1b , F(3,20) = 11.88, p = 0.0001 for Tnf ), and unpaired two-tailed Student’s t test ( f ). g – i HEK293T cells were transfected with a luciferase vector that expresses luciferase under the promoter containing NF-κB-binding elements, and various TLRs. Then, cells were treated h α-Syn protein with indicated concentration ( g ( n = 9), h ( n = 9–12), i ( n = 9)), 50 ng/ml LPS ( i ), or Pam 3 CSK 4 ( i ) for 24 h without ( g , h ) or with ( i ) 10 µg/ml TLR2-blocking antibody (T2.5) and control IgG. p -values were calculated by two-way ANOVA with Bonferroni post hoc test. In g , F(4,80) = 19.63, p = 2.74E-11 for interaction; F(4,80) = 27.31, p = 2.66E-14 for hα-Syn; F(1,80) = 47.95, p = 9.90E-10 for protein overexpression. In h , F(5,126) = 6.070, p = 4.54E-05 for interaction; F(5,126) =6.070, p = 4.54E-05 for hα-Syn; F(1,126) = 73.83, p = 2.77E-14 for protein overexpression. In ( i ), F(3,64) = 16.98, p = 3.19E-08 for interaction; F(3,64) = 177.4, p = 5.90E-31 for stimuli; F(1,64) = 17.47, p = 9.03E-05 for antibody. Data are representative of at least three independent experiments. All values are reported as mean ± SEM.

Article Snippet: Cells were transfected using Lipofectamine ® 2000 (Invitrogen) with pcDNA3 vectors encoding either TLR1 (Addgene #13014), TLR2 (Addgene #13015), TLR4 (Addgene #13018), MD2 (Addgene #13028), CD14 (Addgene #13645), TLR5 (Addgene #13019), or TLR6 (Addgene #13020) with pGL4.32[luc2P/NF-κB-RE/Hygro] vector containing five copies of an NF-κB response element (NF-κB-RE) that drives transcription of the luciferase reporter gene luc2P ( Photinus pyralis ) (#N111A, Promega, Madison, WI).

Techniques: Immunostaining, Ubiquitin Proteomics, Concentration Assay, Blocking Assay, Quantitative RT-PCR, Two Tailed Test, Transfection, Luciferase, Plasmid Preparation, Binding Assay, Control, Over Expression

Journal: Canadian Respiratory Journal

Article Title: PM2.5 Induced the Expression of Fibrogenic Mediators via HMGB1-RAGE Signaling in Human Airway Epithelial Cells

doi: 10.1155/2018/1817398

Figure Lengend Snippet: PM2.5 induces HMGB1 secretion and upregulates RAGE expression in HBECs. HBECs were incubated with PM2.5 for 12, 24, and 48 h. (a) Based on the ELISA results, HMGB1 secretion was increased upon stimulation with PM2.5 (20 μ g/ml) for 24 and 48 h; no significant changes were observed at 12 h. (b) ELISA results show that HMGB1 expression increases 48 h after PM2.5 exposure in a concentration-dependent manner. (c) Real-time quantitative PCR analysis showing an increase in the expression of the RAGE mRNA after 48 h of exposure to PM2.5 (20 μ g/ml); no significant differences were observed in the levels of the TLR2 and TLR4 mRNAs. These data show 1.48-fold (RAGE), 1.14-fold (TLR2), and 1.12-fold (TLR4) increases compared with the untreated control, respectively, using the GAPDH mRNA for calibration. (d) Western blot analysis showing that PM2.5 (20 μ g/ml) increases the levels of the RAGE protein, with no significant changes in the levels of the TLR2 and TLR4 proteins. (e) Immunofluorescence staining shows that PM2.5 (20 μ g/ml) affects the levels of the RAGE protein. The RAGE/GAPDH ratio in control cells is set to 1. # P < 0.05, compared with the control group. ※ P < 0.05, compared with the PM2.5 group, n = 3.

Article Snippet: The anti-TLR2 antibody was purchased from Novus Biologicals (Novus, CO, USA).

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Real-time Polymerase Chain Reaction, Control, Western Blot, Immunofluorescence, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The A subunit of type IIb enterotoxin (LT-IIb) suppresses the proinflammatory potential of the B subunit and its ability to recruit and interact with TLR2.

doi: 10.4049/jimmunol.178.8.4811

Figure Lengend Snippet: FIGURE 3. LT-IIb antagonizes TLR2/1-dependent NF-B activation by LT-IIb-B5 through a cAMP/PKA mechanism. HEK-293 cells cotrans- fected with TLR2/TLR1 and NF-B reporter system were exposed to 2 g/ml LT-IIb-B5, LT-IIb, or a combination of the two, with or without 30-min pretreatment with SQ22536 (200 M) or with H89 (10 M). Fol- lowing a 6-h stimulation period, NF-B-dependent transcription of a lu- ciferase reporter gene was determined as relative luciferase activity (RLA) normalized to that of unstimulated cells. Results are presented as mean SD (n 3) from one of two experiments that yielded similar findings. Statistically significant (, p 0.05) inhibition of LT-IIb-B5-induced cell activation in the presence of LT-IIb and significant (F, p 0.05) reversal of the LT-IIb effect are indicated.

Article Snippet: Because recombinant TLR2 was expressed as a fusion protein with the Fc region of human IgG (R&D Systems), binding to recombinant CD14 (which is similarly fused to the Fc region of human IgG) was used as a negative control.

Techniques: Activation Assay, Luciferase, Activity Assay, Inhibition

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The A subunit of type IIb enterotoxin (LT-IIb) suppresses the proinflammatory potential of the B subunit and its ability to recruit and interact with TLR2.

doi: 10.4049/jimmunol.178.8.4811

Figure Lengend Snippet: FIGURE 4. Differential abilities of LT-IIb and LT-IIb-B5 for inducing TLR2/1-ganglioside associations. Human monocytes were untreated or pretreated for 30 min with 10 mM MCD and then stimulated with LT-IIb or LT-IIb-B5. FRET was used to measure energy transfer between the indicated receptors and GM1 (A) or GD1a (B). Results are shown as the mean SD of the percentage of energy transfer, calculated from three independent experiments. The maximum and minimum energy transfer efficiencies in the system were 37 1.5 and 4 1, respectively (data not shown), and were determined as the energy transfer between two different epitopes on the same molecule (CD14) (maximum) or between molecules that do not engage in heterotypic associations (CD14 and MHC class I) (minimum). Significantly higher activities (, p 0.05) by LT-IIb-B5 com- pared with LT-IIb are indicated. Significant reversal (F, p 0.05) of energy transfer increase due to MCD pretreatment is shown.

Article Snippet: Because recombinant TLR2 was expressed as a fusion protein with the Fc region of human IgG (R&D Systems), binding to recombinant CD14 (which is similarly fused to the Fc region of human IgG) was used as a negative control.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The A subunit of type IIb enterotoxin (LT-IIb) suppresses the proinflammatory potential of the B subunit and its ability to recruit and interact with TLR2.

doi: 10.4049/jimmunol.178.8.4811

Figure Lengend Snippet: FIGURE 5. Differential requirement for TLR2 in cytokine induction by LT-IIb and LT-IIb-B5. Mouse macrophages from wild-type (WT) mice or mice deficient in TLR2, TLR4, or both TLR2 and TLR4 (TLR2/4) were stimulated for 16 h with LT-IIb and LT-IIb-B5 (2 g/ml). Induction of IL-10 (A and B) or IL-6 (C and D) release in culture supernatants was assayed by ELISA. Results are presented as mean SD (n 3) from one of two independent sets of experiments yielding similar findings. Statisti- cally significant (, p 0.05) inhibition of cytokine release in TLR-defi- cient cells compared with corresponding wild-type controls is indicated.

Article Snippet: Because recombinant TLR2 was expressed as a fusion protein with the Fc region of human IgG (R&D Systems), binding to recombinant CD14 (which is similarly fused to the Fc region of human IgG) was used as a negative control.

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The A subunit of type IIb enterotoxin (LT-IIb) suppresses the proinflammatory potential of the B subunit and its ability to recruit and interact with TLR2.

doi: 10.4049/jimmunol.178.8.4811

Figure Lengend Snippet: FIGURE 6. Induction of cAMP production by wild-type LT-IIb, cata- lytic mutants, and LT-IIb-B5. A, RAW264.7 mouse macrophages were stimulated with forskolin (20 M), wild-type LT-IIb, single or dual point catalytic mutants (S59K and S59K/E110K), or LT-IIb-B5 (all at 1 g/ml) and assayed for induction of intracellular cAMP levels using a cAMP en- zyme immunoassay kit (Cayman Chemicals). B, LT-IIb-B5-stimulated macrophages were assayed for intracellular cAMP as above, and also for induction of PGE2 in culture supernatants by ELISA. Before LT-IIb-B5 stimulation or treatment with medium only (M), the cells were pretreated with indomethacin (IND, 1 M) to determine whether inhibition of PGE2 synthesis affects cAMP induction. TLR2-dependence of LT-IIb-B5-in- duced PGE2 (inset) using wild-type and TLR2-deficient macrophages was determined. Results are presented as mean SD (n 3) from typical experiments. Statistically significant decrease of cAMP induction com- pared with wild-type LT-IIb (, p 0.05) or compared with the single point mutant (S59K) (F, p 0.05) is shown. Significant induction of cAMP or PGE2 (, p 0.05) compared with medium-treated cells and significant reversal of these activities by indomethacin (F, p 0.05) are shown.

Article Snippet: Because recombinant TLR2 was expressed as a fusion protein with the Fc region of human IgG (R&D Systems), binding to recombinant CD14 (which is similarly fused to the Fc region of human IgG) was used as a negative control.

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition, Mutagenesis

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The A subunit of type IIb enterotoxin (LT-IIb) suppresses the proinflammatory potential of the B subunit and its ability to recruit and interact with TLR2.

doi: 10.4049/jimmunol.178.8.4811

Figure Lengend Snippet: FIGURE 9. Differential abilities of wild-type or mutant LT-IIb and LT- IIb-B5 for TLR2 binding. A, Binding of LT-IIb or LT-IIb-B5 (10 g/ml) to the indicated receptors was determined on receptor-coated microtiter wells probed with anti-LT-IIb Ab. Following addition of peroxidase-conjugated secondary Ab, binding was measured colorimetrically. B, Binding of bio- tinylated LT-IIb-B5 (10 g/ml) to plate-bound TLR2 in the presence of excess unlabeled LT-IIb-B5 at the indicated concentrations. Bound protein was probed with peroxidase-conjugated streptavidin. C, CHO-K1 cells were transfected with empty vector or plasmids encoding human TLR2 or TLR4 (coexpressed with MD2). The cells were incubated with biotinylated LT-IIb-B5 (1 g/ml), in the presence or absence of anti-TLR2 mAb or IgG2a isotype control (10 g/ml), or 100-fold excess of unlabeled LT- IIb-B5 or LT-IIb, and binding was measured as cell-associated fluorescence in relative fluorescence units (RFU) after staining with FITC-conjugated streptavidin. Data are shown as mean SD of triplicate determinations from typical experiments with similar findings. Significantly higher LT- IIb-B5 binding (, p 0.05) compared with wild-type or mutant LT-IIb is shown in A. Significant (, p 0.05) inhibition of binding is indicated in B. In C, significant binding compared with vector-transfected cells (, p 0.05), and significant reversal of binding (F, p 0.05) are shown.

Article Snippet: Because recombinant TLR2 was expressed as a fusion protein with the Fc region of human IgG (R&D Systems), binding to recombinant CD14 (which is similarly fused to the Fc region of human IgG) was used as a negative control.

Techniques: Mutagenesis, Binding Assay, Transfection, Plasmid Preparation, Incubation, Control, Staining, Inhibition

Journal: Shock

Article Title: Pulmonary Contusion Is Associated With Toll-Like Receptor 4 Upregulation and Decreased Susceptibility to Pseudomonas Pneumonia in a Mouse Model

doi: 10.1097/shk.0b013e31824ee551

Figure Lengend Snippet: FIG. 6. Percentage of surface TLR 4+ and TLR 2+ expression on macrophages and granulocytes in shams and pulmonary-contused mice. Lung tissues were harvested, homogenized, and stained using the methods described. A, Percentage of GR-1+ expressing TLR2 was significantly higher in the PC group vs. sham (*P = 0.0038 using unpaired t test). B, There was no difference in percentage of F4/80+ expressing TLR2 in the PC group vs. sham. C, There was no difference in percentage of GR-1+ expressing TLR4 in the PC group vs. sham. D, Percentage of F4/80+ expressing TLR4 was significantly elevated in the PC group vs. sham (*P G 0.0001 using unpaired t test).

Article Snippet: Cells were then washed, counted and stained with GR-1, F4/80, and either TLR2 or TLR4 antibodies (Imgenex, San Diego, Calif; monoclonal antibody against TLR2/ CD282 fluorescein isothiocyanate conjugate, monoclonal antibody to human TLR4/CD284 fluorescein isothiocyanate conjugate), and acquired on a FACScan flow cytometer (Becton Dickinson).

Techniques: Expressing, Staining

Journal: Shock

Article Title: Pulmonary Contusion Is Associated With Toll-Like Receptor 4 Upregulation and Decreased Susceptibility to Pseudomonas Pneumonia in a Mouse Model

doi: 10.1097/shk.0b013e31824ee551

Figure Lengend Snippet: FIG. 7. Flow cytometric analysis of lung cells. A, Dot-plot histogram of GR-1 F4/80 for a control animal. B, Dot-plot histogram of GR-1 F4/80 for a PC animal. The F4/80+ GR-1hi population in the circle appears in PC mice but not in control mice. Whether this population is included in the analysis, F4/ 80+ cells are still significantly more positive for TLR4 (PC:CTL, 40.97 T 2.215:10.20 T 0.8688 [P G 0.0001], including GR-1hi vs. 40.28 T 2.271:8.183 T 0.7246 [P G 0.0001] without GR-1hi). C and D, The filled gray region repre- sents isotype control; the thin line represents a sample control animal; the thick line represents a sample PC animal. C, F4/80+ cells measured for TLR4 (left) and TLR2 (right). D, GR-1+ F4/80j cells measured for TLR4 (left) and TLR2 (right).

Article Snippet: Cells were then washed, counted and stained with GR-1, F4/80, and either TLR2 or TLR4 antibodies (Imgenex, San Diego, Calif; monoclonal antibody against TLR2/ CD282 fluorescein isothiocyanate conjugate, monoclonal antibody to human TLR4/CD284 fluorescein isothiocyanate conjugate), and acquired on a FACScan flow cytometer (Becton Dickinson).

Techniques: Control

Journal: Journal of Neuroscience

Article Title: Morphine Modulation of Toll-Like Receptors in Microglial Cells Potentiates Neuropathogenesis in a HIV-1 Model of Coinfection with Pneumococcal Pneumoniae

doi: 10.1523/jneurosci.0870-12.2012

Figure Lengend Snippet: Figure10. MorphineTATS.pneumoniae(luciferasetagged)inducedsynergisticincreaseinbacterialdisseminationand proinflammatorycytokinesaresignificantlyattenuatedinTLR2,4knock-outsandTLR2/4doubleknock-outmice.A,Asignificant decrease in bacterial dissemination into the CNS of TLR 2, 4 knock-outs and TLR2/4 double knock-out mice following morphine, TAT, and S. pneumoniae lysate treatment. B, Quantification of bacterial dissemination in the brain tissue of WT, TLR2KO, and TLR4KO mice. Data represents mean SEM of three independent experiments. *p 0.01, ***p 0.001. C, To determine apoptosis,animals(6pergroup)weretreatedasdescribedinFigure4Aandkilledat72h.Brainswereremovedandsnapfrozenin liquid nitrogen. Cryostat sections (5 m) were used to evaluate apoptosis using TUNEL staining (Intergen) according to the manufacturer’s instruction. DAPI staining shows the nuclei of cells. Apoptosis is significantly lower in the TLR knock-out animals compared with wild-type following treatment with morphine, TAT, and S. pneumoniae (S.p.) lysate. Scale bars, 10 m. D, Synergistic increase in proinflammatory cytokines in WT mice following morphine, TAT, and S. pneumoniae lysate treatment is significantly attenuated in brain homogenate harvested from TLR 2, 4 knock-outs and TLR2/4 double knock-out mice. Data representsmeanSEMofthreeindependentexperiments.*p0.01.E,SurvivalcurveofWTandTLR2,4knock-outsandTLR2/4 double knock-out mice treated with morphine TAT S. pneumoniae. Animals were treated as described in Figure 4A and survival followed for 5 d. Data represents mean SEM of three independent experiments.

Article Snippet: Primer sequences used in the amplification of TLR genes are TLR4 (accession no. NM_021297), TLR2 (accession no. NM_011905), and TLR9 (accession no. NM_031178) (all from Origene Techonologies).

Techniques: Knock-Out, TUNEL Assay, Staining