Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: Primer used for Real-time qPCR.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Sequencing

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: Cell surface level of TLR4 in BV-2 cells treated with chestnut leaf or spiny bur extracts and LPS. BV-2 cells were pre-treated (or not) with spiny bur (SB) or leaf (L) extracts (0.5 mg/mL) for 3 h, incubated with or without LPS (0.5 μg/mL) for a total of 24 h, then subjected to flow cytometric analysis of the immunostaining for TLR4 receptor. ( a ) Representative plots of untreated BV-2 cells ( b ) Representative plots of BV-2 cells not activated with LPS ( c ) Representative plots of LPS-stimulated BV-2 cells ( d ) Relative quantification is expressed as MFI, median fluorescence intensity. Results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. ° p < 0.05 significantly different from control cells; * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Incubation, Immunostaining, Fluorescence, Control

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: RT-PCR analysis of TLR4 gene expression in BV-2 cells treated with chestnut extracts. BV-2 cells were treated with spiny bur or leaf extracts (0.5 mg/mL) for 3 h, then stimulated (or not) with LPS (0.5 µg/mL) for a total of 24 h. At the end of incubation, RNA was extracted from cells and samples were subjected to RT-PCR analysis using a specific primer for TLR4, as explained in the Materials and Methods section. The relative mRNA content of TLR4 was normalised to ( a ) untreated control and ( b ) untreated LPS control. Relative quantification is obtained as reported in the Materials and Methods section; results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. ° p < 0.05 significantly different from control cells; * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Control

Journal: Antioxidants

Article Title: By-Product Extracts from Castanea sativa Counteract Hallmarks of Neuroinflammation in a Microglial Model

doi: 10.3390/antiox12040808

Figure Lengend Snippet: RT-PCR analysis of TLR4 and CD14 gene expression in BV-2 cells treated with different chestnut leaf extract fractions. BV-2 cells were treated for 3 h with 50 μg/mL of fractions at different polarity (L Fr.), then stimulated with 0.5 μg/mL LPS for a total of 24 h. At the end of incubation, RNA was extracted from cells and samples were subjected to RT-PCR analysis using a specific primer for TLR4 ( a ) and CD14 ( b ), as reported in the Materials and Methods section. The relative mRNA contents of TLR4 and CD14 were normalised to LPS-treated cells (red bars). Relative quantification is obtained as described in the Materials and Methods section, and results are expressed as means ± SEM of three independent experiments. Statistical analysis was performed by Fisher’s LSD test following one-way ANOVA. * p < 0.05 significantly different from LPS-treated cells.

Article Snippet: Rabbit anti-TLR4 polyclonal FITC-conjugated antibody was purchased from StressMarq.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation

Journal: Frontiers in Immunology

Article Title: Lactoferrin Retargets Human Adenoviruses to TLR4 to Induce an Abortive NLRP3-Associated Pyroptotic Response in Human Phagocytes

doi: 10.3389/fimmu.2021.685218

Figure Lengend Snippet: TLR4 engagement and signalling involved in HAdV-lactoferrin DC uptake and maturation. (A) HAdV-lactoferrin complexes were incubated with recombinant TLR4/MD-2 for 30 min. and then added to DCs. Uptake was quantified 24 h post-incubation by flow cytometry (n =11, statistical analyses by two-tailed paired t-test); (B) DCs were pre-treated for 1 h with TAK-242, HAdV-lactoferrin complex uptake was analysed 24 h post-incubation by flow cytometry (n = 6, statistical analyses by two-tailed paired t-test); (C) IL-1β release following pre-treatment with TAK-242 (n ≥ 3 statistical analyses by t-test); (D) TNF levels 24 h post-incubation ± TAK-242 (n ≥ 3, statistical analyses by two-tailed paired t-test); (E) Percent infection following inhibition with Pepinh-TRIF (n ≥ 6, statistical analyses by two-tailed paired t-test); (F) Percent infection following inhibition with oxPAPC (n ≥ 6, statistical analyses by two-tailed paired t-test); (G) IL-1β release from DCs incubated with HAdV-lactoferrin ± oxPAPC and Pepinh-TRIF (n = 3, statistical analyses by two-tailed paired t-test).

Article Snippet: Fluorescence intensity was assessed at 24 h. TLR4 surface expression level was assessed with an anti-TLR4 antibody (Miltenyi Biotech) after 4 or 24 h. DC maturation at 24 h post-incubation was assessed by measuring CD86 surface expression level with an anti-CD86 antibody (clone 2331, BD Biosciences) or by measuring dextran (4.4 kDa) uptake (dextran TRITC, Sigma).

Techniques: Incubation, Recombinant, Flow Cytometry, Two Tailed Test, Infection, Inhibition

Journal: Frontiers in Immunology

Article Title: Lactoferrin Retargets Human Adenoviruses to TLR4 to Induce an Abortive NLRP3-Associated Pyroptotic Response in Human Phagocytes

doi: 10.3389/fimmu.2021.685218

Figure Lengend Snippet: TLR4-mediated HAdV-lactoferrin uptake in DCs and IL-1β release Lactoferrin binds to HAdV capsid and retargets the capsid toward TLR4 complex on the cells surface. Following TLR4 engagement, its TIR domain recruits MyD88 and TIRAP, which bridge TLRs to IRAK and MAPK family members that activate NF-kB, AP-1, and IRF. This priming event initiates transcription of genes coding for inflammasome components (e.g., NLRP3 and IL-1β). Under prototypic conditions, DCs detect a second perturbation (signal 2) that induced ROS release (mitochondrial stress) or K + efflux (perturbations of cellular integrity), and/or cathepsin B release from lysosome rupture. These pathways do not appear to be activated by HAdV-lactoferrin complexes. In addition to TLR4 pathways, the RIPK1-RIPK3 pathway is activated through an autocrine-TNF release. During inflammasome formation, pro-caspase-1 auto-activation induces cleavage of pro-IL-1β and likely GSDMD, which will initiate, but not complete, the loss of plasma membrane integrity via pore formation, allowing IL-1β release. Twenty-four hours post-challenge, DC membrane integrity is intact, consistent with the involvement of ESCRT-III complex and repairing GSDMD pores.

Article Snippet: Fluorescence intensity was assessed at 24 h. TLR4 surface expression level was assessed with an anti-TLR4 antibody (Miltenyi Biotech) after 4 or 24 h. DC maturation at 24 h post-incubation was assessed by measuring CD86 surface expression level with an anti-CD86 antibody (clone 2331, BD Biosciences) or by measuring dextran (4.4 kDa) uptake (dextran TRITC, Sigma).

Techniques: Activation Assay, Clinical Proteomics, Membrane