Journal: Frontiers in Immunology

Article Title: Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response

doi: 10.3389/fimmu.2023.1187035

Figure Lengend Snippet: EV71 infection activates the antiviral innate immunity via toll-like receptor (TLR) monomers and TLR2 heterodimers to limit its replication. (A) Human-derived TLR2 and TLR2 heterodimer (TLR2/TLR1 and TLR2/TLR6) or (B) mouse-derived TLR2 heterodimer (mTLR2/mTLR1 and mTLR2/mTLR6) plasmids were transfected into HEK293 cells at a dose of 1 or 2 μg for 24 h, followed by infection with EV71 at a multiplicity of infection (MOI) of 1 for 24 (h) n = 3. (C) Human-derived TLR1, TLR6, and TLR2/CD14 or (D) mouse-derived mTLR2, mTLR1, and mTLR6 plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) (E) Human-derived TLR2 and TLR2 heterodimer (TLR2/TLR1 and TLR2/TLR6) plasmids were transfected into rhabdomyosarcoma (RD) cells for 24 h and infected with EV71 at an MOI of 0.5 or 1 for 24 (h) Genome copies of EV71 were determined using quantitative polymerase chain reaction (qPCR), and relative fold differences compared with HEK293 cells infected with EV71 for 24 h were calculated. n = 3. (F) Human-derived TLR2 heterodimer (TLR2/TLR1, TLR2/TLR6, and TLR2/CD14) or (G) mouse-derived TLR2 heterodimer (mTLR2/mTLR1 and mTLR2/mTLR6) plasmids were transfected into HEK293 cells at a dose of 1 or 2 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) Supernatants were collected and interleukin (IL)-8 concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) kit. n = 3. (H) Human-derived TLR monomer (TLR2, TLR1, and TLR6) and TLR2 heterodimer (TLR2/TLR1, TLR2/TLR6, and TLR2/CD14) plasmids or (I) mouse-derived TLR monomer (mTLR2, mTLR1, and mTLR6) and TLR2 heterodimer (mTLR2/mTLR1 and mTLR2/mTLR6) plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) Total proteins were collected, and activation of the phosphoinositide 3-kinase/protein kinase B ( PI3K/AKT ) and mitogen-activated protein kinase ( MAPK ) pathways was assessed via western blotting. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: All these plasmids were purchased from Addgene ( https://www.addgene.org ). mTLR6 plasmid pUNO1-mTLR06-HA3x (puno1ha-mtlr6), TLR2 heterodimer plasmids, including pDUO-hTLR6/TLR2 (pduo-htlr6tlr2), pDUO-hTLR1/TLR2 (pduo-htlr1tlr2), and pDUO-hCD14/TLR2 (pduo-hcd14tlr2) plasmids, TLR1/2/4/6 Dominant-negative TIR-less (DN [ΔTIR]) plasmids, pUNO1-hTLR1-DN-HA (puno1ha-htlr1-dn), pUNO1-hTLR2-DN-HA (puno1ha-htlr2-dn), pUNO1-hTLR4-DN-HA (puno1ha-htlr4a-dn), and pUNO1-hTLR6-DN-HA (puno1ha-htlr6-dn), were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Infection, Derivative Assay, Transfection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response

doi: 10.3389/fimmu.2023.1187035

Figure Lengend Snippet: EV71 replication is inhibited by conditional changes in TLR2 heterodimers. (A) Human–mouse chimeric TLR2 heterodimer (mTLR2/TLR1, TLR2/mTLR1, mTLR2/TLR6, and TLR2/mTLR6) plasmids and (B) single dominant-negative TIR-less (DN) TLR2 heterodimer (DN-TLR2/TLR1, TLR2/DN-TLR1, DN-TLR2/TLR6, and TLR2/DN-TLR6) plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) DN-TLR (DN-TLR1, DN-TLR2, and DN-TLR6) plasmids were transfected into (C) HEK293 or (D) UT-SCC-60B cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. Genome copies of EV71 were determined and relative fold differences were calculated. (E) Human–mouse chimeric TLR2 heterodimer (mTLR2/TLR1, TLR2/mTLR1, mTLR2/TLR6, and TLR2/mTLR6) plasmids were transfected into HEK293 cells at a dose of 1 or 2 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. (F) Concentrations of IL-8 in the supernatant of group (B) . n = 3. (G) Human–mouse chimeric TLR2 heterodimer (mTLR2/TLR1, TLR2/mTLR1, mTLR2/TLR6, and TLR2/mTLR6) plasmids or (H) single dominant-negative TIR-less TLR2 heterodimer (DN-TLR2/TLR1 and TLR2/DN-TLR1) plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) Total proteins were collected, and activation of the PI3K/AKT and MAPK pathways was assessed via western blotting. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: All these plasmids were purchased from Addgene ( https://www.addgene.org ). mTLR6 plasmid pUNO1-mTLR06-HA3x (puno1ha-mtlr6), TLR2 heterodimer plasmids, including pDUO-hTLR6/TLR2 (pduo-htlr6tlr2), pDUO-hTLR1/TLR2 (pduo-htlr1tlr2), and pDUO-hCD14/TLR2 (pduo-hcd14tlr2) plasmids, TLR1/2/4/6 Dominant-negative TIR-less (DN [ΔTIR]) plasmids, pUNO1-hTLR1-DN-HA (puno1ha-htlr1-dn), pUNO1-hTLR2-DN-HA (puno1ha-htlr2-dn), pUNO1-hTLR4-DN-HA (puno1ha-htlr4a-dn), and pUNO1-hTLR6-DN-HA (puno1ha-htlr6-dn), were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Dominant Negative Mutation, Transfection, Infection, Activation Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response

doi: 10.3389/fimmu.2023.1187035

Figure Lengend Snippet: EV71 infection activates antiviral innate immunity via TLR4 and TLR2/TLR4 to limit its replication. (A) Human-derived TLR4 and TLR2/TLR4 plasmids or (B) mouse-derived mTLR4 and mTLR2/mTLR4 plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. (C) Human-derived TLR4 and TLR2/TLR4 plasmids were transfected into RD cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 0.5 or 1 for 24 (h) n = 3. Genome copies of EV71 were determined and relative fold differences were calculated. (D) Human-derived TLR4 and TLR2/TLR4 plasmids were transfected into HEK293 cells at a dose of 1 or 2 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. (E) Concentrations of IL-8 in the supernatant of group (B) were determined. (F) Human-derived TLR2, TLR4, and TLR2/TLR4 plasmids or (G) mouse-derived mTLR2, mTLR4, and mTLR2/mTLR4 plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. Total proteins were collected, and activation of the PI3K/AKT and MAPK pathways was assessed via western blotting. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: All these plasmids were purchased from Addgene ( https://www.addgene.org ). mTLR6 plasmid pUNO1-mTLR06-HA3x (puno1ha-mtlr6), TLR2 heterodimer plasmids, including pDUO-hTLR6/TLR2 (pduo-htlr6tlr2), pDUO-hTLR1/TLR2 (pduo-htlr1tlr2), and pDUO-hCD14/TLR2 (pduo-hcd14tlr2) plasmids, TLR1/2/4/6 Dominant-negative TIR-less (DN [ΔTIR]) plasmids, pUNO1-hTLR1-DN-HA (puno1ha-htlr1-dn), pUNO1-hTLR2-DN-HA (puno1ha-htlr2-dn), pUNO1-hTLR4-DN-HA (puno1ha-htlr4a-dn), and pUNO1-hTLR6-DN-HA (puno1ha-htlr6-dn), were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Infection, Derivative Assay, Transfection, Activation Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response

doi: 10.3389/fimmu.2023.1187035

Figure Lengend Snippet: Conditional changes in TLR4 and TLR2/TLR4 heterodimers inhibit EV71 replication by activating innate immunity. (A) Human–mouse chimeric TLR2 heterodimer (mTLR2/TLR4 and TLR2/mTLR4) plasmids were transfected into HEK293 cells at a dose of 1 or 2 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. (B) Single dominant-negative TIR-less TLR2/TLR4 heterodimer (DN-TLR2/TLR4 and TLR2/DN-TLR4) plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) Single dominant-negative TIR-less TLR4 plasmid was transfected into (C) HEK293 or (D) UT-SCC-60B cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. (E) TLR4 single nucleotide mutation plasmids (A896G, C1196T, and C2141A) were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by infection with EV71 at an MOI of 1 for 24 (h) n = 3. Genome copies of EV71 were determined and relative fold differences were calculated. Concentrations of IL-8 in the supernatants of (F) human–mouse chimeric TLR2 heterodimer and (G) single dominant-negative TIR-less TLR2 heterodimer groups were determined. n = 3. Activation of the PI3K/AKT and MAPK pathways in (H) human–mouse chimeric TLR2 heterodimers and (I) DN-TLR2/TLR4 groups was assessed via western blotting. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: All these plasmids were purchased from Addgene ( https://www.addgene.org ). mTLR6 plasmid pUNO1-mTLR06-HA3x (puno1ha-mtlr6), TLR2 heterodimer plasmids, including pDUO-hTLR6/TLR2 (pduo-htlr6tlr2), pDUO-hTLR1/TLR2 (pduo-htlr1tlr2), and pDUO-hCD14/TLR2 (pduo-hcd14tlr2) plasmids, TLR1/2/4/6 Dominant-negative TIR-less (DN [ΔTIR]) plasmids, pUNO1-hTLR1-DN-HA (puno1ha-htlr1-dn), pUNO1-hTLR2-DN-HA (puno1ha-htlr2-dn), pUNO1-hTLR4-DN-HA (puno1ha-htlr4a-dn), and pUNO1-hTLR6-DN-HA (puno1ha-htlr6-dn), were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Transfection, Infection, Dominant Negative Mutation, Plasmid Preparation, Mutagenesis, Activation Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response

doi: 10.3389/fimmu.2023.1187035

Figure Lengend Snippet: EV71 capsid proteins induce cytokine responses via TLR2 and TLR2 heterodimers. UT-SCC-60B cells were stimulated with purified recombinant prokaryotic-expressed EV71 capsid proteins at a final concentration of 80 μg/mL, or EV71 capsid recombinant eukaryotic plasmids were transfected into UT-SCC-60B cells at a dose of 1 μg for 24 (h) n = 3. Concentrations of IL-6 and IL-8 in (A) recombinant EV71 capsid protein stimulated and (B) EV71 capsid plasmid transfected groups were determined. Activation of the PI3K/AKT and MAPK pathways in (C) recombinant EV71 capsid protein stimulated and (D) EV71 capsid plasmid transfected groups were assessed via western blotting. Human-derived TLR2 and TLR2 heterodimer (TLR2/TLR1 and TLR2/TLR6) plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h and stimulated with recombinant EV71 capsid proteins at a final concentration of 80 μg/mL for 24 (h) n = 3. (E) Concentrations of IL-8 and (G) activation of the PI3K/AKT and MAPK pathways were determined. Human-derived TLR2 and TLR2 heterodimer (TLR2/TLR1 and TLR2/TLR6) and EV71 capsid plasmids were co-transfected into UT-SCC-60B cells at a dose of 1 μg for 24 (h) n = 3. (F) Concentrations of IL-8 and (H) activation of the PI3K/AKT and MAPK pathways were determined. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: All these plasmids were purchased from Addgene ( https://www.addgene.org ). mTLR6 plasmid pUNO1-mTLR06-HA3x (puno1ha-mtlr6), TLR2 heterodimer plasmids, including pDUO-hTLR6/TLR2 (pduo-htlr6tlr2), pDUO-hTLR1/TLR2 (pduo-htlr1tlr2), and pDUO-hCD14/TLR2 (pduo-hcd14tlr2) plasmids, TLR1/2/4/6 Dominant-negative TIR-less (DN [ΔTIR]) plasmids, pUNO1-hTLR1-DN-HA (puno1ha-htlr1-dn), pUNO1-hTLR2-DN-HA (puno1ha-htlr2-dn), pUNO1-hTLR4-DN-HA (puno1ha-htlr4a-dn), and pUNO1-hTLR6-DN-HA (puno1ha-htlr6-dn), were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Purification, Recombinant, Concentration Assay, Transfection, Plasmid Preparation, Activation Assay, Western Blot, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Cell membrane-bound toll-like receptor-1/2/4/6 monomers and -2 heterodimer inhibit enterovirus 71 replication by activating the antiviral innate response

doi: 10.3389/fimmu.2023.1187035

Figure Lengend Snippet: EV71 capsid proteins induce cytokine responses via TLR4 and TLR2/TLR4 heterodimer. Human-derived TLR4 and TLR2/TLR4 plasmids were transfected into HEK293 cells at a dose of 1 μg for 24 h, followed by stimulation with recombinant EV71 capsid proteins at a final concentration of 80 μg/mL for 24 (h) n = 3. (A) Concentrations of IL-8 and (C) activation of the PI3K/AKT and MAPK pathways were determined. Human-derived TLR4 and TLR2/TLR4 and EV71 capsid plasmids were co-transfected into UT-SCC-60B cells at a dose of 1 μg for 24 (h) n = 3. (B) Concentrations of IL-8 and (D) activation of the PI3K/AKT and MAPK pathways were determined. (E) Schematic model for the activation of innate immunity by EV71 and capsid proteins via cell membrane-bound TLR1/2/4/6 monomers and TLR2 heterodimers. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: All these plasmids were purchased from Addgene ( https://www.addgene.org ). mTLR6 plasmid pUNO1-mTLR06-HA3x (puno1ha-mtlr6), TLR2 heterodimer plasmids, including pDUO-hTLR6/TLR2 (pduo-htlr6tlr2), pDUO-hTLR1/TLR2 (pduo-htlr1tlr2), and pDUO-hCD14/TLR2 (pduo-hcd14tlr2) plasmids, TLR1/2/4/6 Dominant-negative TIR-less (DN [ΔTIR]) plasmids, pUNO1-hTLR1-DN-HA (puno1ha-htlr1-dn), pUNO1-hTLR2-DN-HA (puno1ha-htlr2-dn), pUNO1-hTLR4-DN-HA (puno1ha-htlr4a-dn), and pUNO1-hTLR6-DN-HA (puno1ha-htlr6-dn), were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Derivative Assay, Transfection, Recombinant, Concentration Assay, Activation Assay, Membrane

Journal: The Journal of Experimental Medicine

Article Title: Helicobacter pylori metabolites exacerbate gastritis through C-type lectin receptors

doi: 10.1084/jem.20200815

Figure Lengend Snippet: Identification of chemical structure of fraction 13-14 as αCAG. (A) HEK-based NF-κB reporter cells expressing TLR2 or TLR4 were stimulated with HPTLC-separated lipid fractions from H. pylori for 20 h. Activation of NF-κB was assessed by monitoring SEAP activity in the supernatants. (B) GC-MS chromatogram of FAMEs and sterol derived from fraction 13-14. (C) MS spectrum of 24.4 min in B. (D) 13 C-NMR chemical shifts of fraction 13-14 (upper panel) were assigned by HSQC (lower panel) and HMBC experiments. (E) NFAT-GFP reporter cells expressing FcRγ + Flag-tagged Mincle mutants generated by site-directed mutagenesis were stimulated with indicated lipids for 20 h. Induction of GFP was analyzed by flow cytometry (upper panels). Cell surface expression of Mincle was detected by anti-Flag mAb (lower panels). Open histograms show staining with isotype control. The spectrum of binding preferences for the mutants was distinct; in particular, the F198A mutation did not affect TDM recognition, while this mutant poorly recognized αCAG. In contrast, S200A and P202A selectively impaired TDM binding. Note that the C197 is considered to be involved in intramolecular disulfide bonds. Data are presented as the mean ± SD of duplicate assays (A and E).

Article Snippet: HEK293-derived reporter cells that stably express an NF-κB–inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene and TLR2 or TLR4 gene were obtained from InvivoGen.

Techniques: Expressing, High Performance Thin Layer Chromatography, Activation Assay, Activity Assay, Gas Chromatography-Mass Spectrometry, Derivative Assay, Generated, Mutagenesis, Flow Cytometry, Staining, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Helicobacter pylori metabolites exacerbate gastritis through C-type lectin receptors

doi: 10.1084/jem.20200815

Figure Lengend Snippet: Nontargeted lipidomics of H. pylori revealed the regulation of immunostimulatory potential by changing lipid composition. (A) Scanning electron micrographs of helical form and coccoid form of H. pylori . Scale bar, 10 µm. (B) 2D map of mass ( m/z of precursor ions) versus liquid chromatography retention time of cholesteryl lipids isolated from helical (left panel) or coccoid (right panel) form of H. pylori . Plots of precursor ions were identified as cholesterol ester (CE), αCG, αCAG, αCPG, and lyso αCPG (detected only in coccoid form). (C) Lipid extracts from helical or coccoid form of H. pylori were analyzed by HPTLC using chloroform/methanol/water (65:25:4, vol/vol/vol) and stained with copper(II) acetate-phosphoric acid (left) or orcinol (right). Open and closed arrowheads denote the origin and the solvent front, respectively. The black arrow indicates αCAG, and the gray arrow indicates the lipid component increasing in coccoid form (Spot C). (D) Peak area of each fatty acid fragment ion of αCAG that is analyzed in . (E–G) BMDCs were stimulated with αCG, αCAG C14:0, C16:0, or C18:0 (0.02, 0.06, 0.2, 0.6, and 2 nmol/well) for 1 d. The concentrations of TNF (E), MIP-2 (F), and IL-6 (G) in the supernatants were determined by ELISA. (H) Reporter cells expressing Mincle + FcRγ were stimulated with αCG, αCAG C14:0, C16:0, or C18:0 (0.016, 0.08, 0.4 and 2 nmol/well) for 20 h and analyzed for GFP expression. (I) Resident peritoneal exudate cells from WT or Card9 −/− mice were stimulated with αCAG C14:0, Spot C (0.02, 0.2, and 2 nmol/well) or LPS. IL-6 production was quantified by ELISA. (J) NFAT-GFP reporter cells expressing FcRγ alone, Mincle + FcRγ or DCAR + FcRγ or HEK-based NF-κB reporter cells expressing TLR2 or TLR4 were stimulated with HPTLC-separated lipids from coccoid H. pylori for 20 h and analyzed for GFP or SEAP expression. (K) Full scan MS spectra of Spot C (upper panel) and MS/MS spectra of m/z 1208.9006 [M+NH 4 ] + (lower panel) in the positive ion mode. Ion peak at m/z 1208.9006 [M + NH 4 ] + is proposed to be cholesteryl α-phosphatidylpyranoside (calculated mass, 1208.9040). (L) Full-scan MS spectra of Spot C (upper panel) and MS/MS spectra of m/z 1189.8623 [M-H] − (lower panel) in the negative ion mode. Ion peak at m/z 1189.8623 [M-H] − is supposed to be cholesteryl α-phosphatidylpyranoside (calculated mass, 1189.8629). (M) 1 H-NMR spectrum (600 MHz, CDCl 3 :CD 3 OD:D 2 O [65:35:5], 298 K) of Spot C. (N) Chemical structure and 1 H-NMR chemical shifts assignment of Spot C. Chemical shifts are given in δ ppm, followed by integration, multiplicity and J in hertz. (O) Peak area of each fatty acid fragment ion of αCPG that is analyzed in . (P) Reporter cells expressing DCAR + FcRγ were stimulated with purified αCPG, synthesized αCPG (C14:0 19c:0) or αCPG amide analogue (0.1, 0.3 and 1 nmol/well) for 20 h and analyzed for GFP expression. Data are presented as the mean ± SD of triplicate (E–G and I) or duplicate (H, J, and P) assays and are representative of two independent experiments. An unpaired two-tailed Student’s t test was used for the statistical analyses. *, P < 0.05; **, P < 0.01.

Article Snippet: HEK293-derived reporter cells that stably express an NF-κB–inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene and TLR2 or TLR4 gene were obtained from InvivoGen.

Techniques: Liquid Chromatography, Isolation, High Performance Thin Layer Chromatography, Staining, Solvent, Enzyme-linked Immunosorbent Assay, Expressing, Tandem Mass Spectroscopy, Purification, Synthesized, Two Tailed Test