Journal: medRxiv

Article Title: A novel UNC93B1 gain-of-function mutation leads to TLR7 and TLR8 hyperactivation and systemic lupus erythematosus

doi: 10.1101/2024.09.11.24313360

Figure Lengend Snippet: (A) NF-κB luciferase assays in HEK293T cells co-transfected TLR7 (left) or TLR8 (right) with different UNC93B1 expression plasmids, treated as indicated (n = 3 biological replicates). Data represent mean ± SEM; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test. EV: empty vector; WT: wild-type UNC93B1; R95L: R95L mutant; HR: H412R mutant. (B) qPCR analysis of IL8 transcription level in HEK293T cells co-transfected TLR7 (left) or TLR8 (right) with different UNC93B1 expression plasmids, treated as indicated (n = 3 biological replicates). Data represent mean ± SEM; ***, P < 0.001; two-tailed unpaired Student’s t test. (C) qPCR analysis of IL8 in HEK293T cells co-transfected TLR3 with different UNC93B1 expression plasmids, treated as indicated (n = 3 biological replicates). Data represent mean ± SEM; two-tailed unpaired Student’s t test. EV: empty vector; WT: wild-type UNC93B1; R95L: R95L mutant; HR: H412R mutant. (D) NF-κB luciferase assay in HEK293T cells co-transfected TLR9 with different UNC93B1 expression plasmids, treated as indicated (n = 3 biological replicates). Data represent mean ± SEM; *, P < 0.05; two-tailed unpaired Student’s t test. WT: wild-type UNC93B1; R95L: R95L mutant. (E) qPCR analysis of IL1B , IL6 , IL8 and TNF transcription levels in THP-1 expressing the WT or R95L mutant UNC93B1 treated with various TLRs agonists (n = 3 biological replicates). Data represent mean ± SEM; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test. (F, G) Intracellular cytokine staining of Tnf in mouse RAW 264.7 expressing the WT or R95L mutant UNC93B1, and treated with various TLRs agonists or inhibitors. (H, I) qPCR analysis of NF-κB (H) and type I IFN (I) pathways related genes transcription in BMDCs from indicated mice treated with various TLRs agonists(n = 5 mice per group). Data represent mean ± SEM; **, P < 0.01; ***, P < 0.001; two-tailed unpaired Student’s t test.

Article Snippet: For western blotting experiments, TLR7 (5632), TLR8 (11886), TLR9 (13674), and GAPDH (5174) antibodies were purchased from Cell Signaling Technology.

Techniques: Luciferase, Transfection, Expressing, Two Tailed Test, Plasmid Preparation, Mutagenesis, Staining

Journal: medRxiv

Article Title: A novel UNC93B1 gain-of-function mutation leads to TLR7 and TLR8 hyperactivation and systemic lupus erythematosus

doi: 10.1101/2024.09.11.24313360

Figure Lengend Snippet: (A) The structure of TLR7 and UNC93B1 complex (PDB: 7CYN) shows H1 helix (residues 91-97) of UNC93B1 directly contacting two loop regions of the C-terminal LRR-CT domain of TLR7. Protein structural analysis was executed with Pymol. (B) Flag-immunoprecipitation and western blot analyses of UNC93B1 and TLR7 (left)/TLR8 (right) interaction in HEK293T cells. (C) TLR8-immunoprecipitation and western blot analyses of UNC93B1 and TLR8 interaction in HEK293T cells. (D) BioID and western blot analyses of UNC93B1 and TLR7 interaction in RAW 264.7 cells. (E) Immunoblots of TLR7 in endosomes and late endosomes/lysosomes from RAW 264.7 cells expressing the WT or R95L mutant UNC93B1. (F, G) Representative immunofluorescent images (F) and quantification of the colocalization between Tlr7 and Lamp1 or Eea1 in BMDMs from the indicated mice (G) (n = 14-18 cells per group). Data represent mean ± SEM; two-tailed unpaired Student’s t test. (H) RNA pull-down and western blot analyses of ssRNA binding affinity of Tlr7 in BMDCs. (I) Graphic model of how R95L mutation affect UNC93B1/TLR axis and downstream signaling.

Article Snippet: For western blotting experiments, TLR7 (5632), TLR8 (11886), TLR9 (13674), and GAPDH (5174) antibodies were purchased from Cell Signaling Technology.

Techniques: Immunoprecipitation, Western Blot, Expressing, Mutagenesis, Two Tailed Test, Binding Assay

Journal: Thorax

Article Title: Human respiratory syncytial virus viroporin SH: a viral recognition pathway used by the host to signal inflammasome activation.

doi: 10.1136/thoraxjnl-2012-202182

Figure Lengend Snippet: Figure 2 TLR4 provides the first or priming signal of activation. Lung epithelial cells were infected with 5 multiplicity of infection (moi) of respiratory syncytial virus and the presence of pro-IL1β by western blotting was examined, as well as the secretion of IL1β and IFNβ using the CBA system. RIG-I expression was knocked down by psiRNA, and the cells were again infected and examined for pro- IL1β, as well as IL1β and IFNβ secretion (A). TLR3 expression was knocked down by psiRNA and the cells were again infected and tested for pro-IL1β, as well as IL1β and IFNβ secretion (B). TLR7 expression was knocked down by psiRNA and the cells were again infected and tested for pro- IL1β, as well as IL1β and IFNβ secretion (C). TLR4 expression was knocked down by psiRNA and the cells were again infected and tested for pro-IL1β, as well as IL1β and IFNβ secretion (D). TLR8 expression was knocked down by psiRNA and the cells were again infected and tested for pro-IL1β, as well as IL1β and IFNβ secretion (E). TLR2 expression was knocked down by psiRNA and the cells were again infected and tested for pro-IL1β, as well as IL1β and IFNβ secretion (F). The data represent the mean of three independent experiments.

Article Snippet: TLR3, TLR9, TLR7 and TLR8 specific antibodies were purchased from Santa Cruz Biotechnology (California, USA).

Techniques: Activation Assay, Infection, Virus, Western Blot, Expressing

Journal: Journal of Inflammation (London, England)

Article Title: Role of toll-like receptors in human iris pigment epithelial cells and their response to pathogen-associated molecular patterns

doi: 10.1186/1476-9255-11-20

Figure Lengend Snippet: Antibodies used for Western blotting

Article Snippet: TLR8 , Mouse IgG 1, k , - , 44C143 , Imgenex , 2.

Techniques: Western Blot, Concentration Assay

Journal: Journal of Inflammation (London, England)

Article Title: Role of toll-like receptors in human iris pigment epithelial cells and their response to pathogen-associated molecular patterns

doi: 10.1186/1476-9255-11-20

Figure Lengend Snippet: TLR specific primers and RT-PCR conditions

Article Snippet: TLR8 , Mouse IgG 1, k , - , 44C143 , Imgenex , 2.

Techniques: Sequencing, Amplification

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phagosomal signaling by Borrelia burgdorferi in human monocytes involves Toll-like receptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated induction of IFN-beta.

doi: 10.1073/pnas.1013776108

Figure Lengend Snippet: Fig. 1. Transcription of IFN-β is abrogated after TLR8 inhibition in human monocytes. (A) TLR8 inhibition by IRS-957 eliminated transcription of IFN-β in Bb-infected monocytes (10:1 multiplicity of infection), whereas a control ODN sequence did not. Transcription of IFN-β in response to LPS was not affected by either treatment. Relative expression refers to ifnb gene ex- pression normalized to gapdh. N.S., not significant; ODN, oligodeoxyr- ibonucleotide. (B) Secretion of NF-κB–dependent cytokines (TNF-α, IL-6, IL- 10, and IL-1β) was diminished on inhibition of TLR8 in human monocytes. Un, uninfected. P values correspond to paired analysis [Mann–Whitney U test (A) and paired t test (B)] from a minimum of four independent experiments. *P < 0.05; **P < 0.01.

Article Snippet: Following different time points of stimulation (0 min, 30 min, 60 min, 90 min, 3 h, and 4 h), slides were fixed with 4% (vol/vol) paraformaldehyde for 10 min at room temperature (RT); permeabilized with 0.2% saponin for 10 min; blocked with Connaught Medical Research Laboratories (CMRL) 10% FCS for 2 h at RT; incubated overnight at 4 °C with a rabbit anti-human IRF7 polyclonal or mouse anti-human IRF3 monoclonal antibody (Santa Cruz Biotechnology, Inc.), a rabbit anti-TLR8 polyclonal antibody (IMGENEX), or a rabbit anti-TLR2 polyclonal antibody (Rockland Immunochemicals) (1:100 dilution for all); and subsequently incubated with Texas RedR-X–conjugated goat anti-rabbit antibody (1:200 dilution; Invitrogen) for 1 h at RT or goat anti-mouse DyLight 594 (Thermo Scientific).

Techniques: Inhibition, Infection, Control, Sequencing, Expressing, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phagosomal signaling by Borrelia burgdorferi in human monocytes involves Toll-like receptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated induction of IFN-beta.

doi: 10.1073/pnas.1013776108

Figure Lengend Snippet: Fig. 2. TLR2 and TLR8 cellular expression. Costaining for TLR2 (green) and TLR8 (red) is shown in unstimulated monocytes and in both infected and uninfected cells (bystander) within 30 min of incubation with Bb (10:1 mul- tiplicity of infection). TLR2 fluorescent signals are visualized in unstimulated and bystander cells, greatly increasing in Bb-infected monocytes. TLR8 signals are of very low intensity in uninfected monocytes and, like TLR2, increase in Bb-infected monocytes. Black arrows point to degraded GFP-Bb. TLR2 coloc- alizes with TLR8 even at locations that do not coincide with internalized GFP- Bb (white arrows and region of interest). Numerical values shown in white correspond to Mander’s colocalization coefficient (M2), indicative of the proportion of the green signal (TLR2) overlapping with the signal in the red channel (TLR8). DIC, differential interference contrast.

Article Snippet: Following different time points of stimulation (0 min, 30 min, 60 min, 90 min, 3 h, and 4 h), slides were fixed with 4% (vol/vol) paraformaldehyde for 10 min at room temperature (RT); permeabilized with 0.2% saponin for 10 min; blocked with Connaught Medical Research Laboratories (CMRL) 10% FCS for 2 h at RT; incubated overnight at 4 °C with a rabbit anti-human IRF7 polyclonal or mouse anti-human IRF3 monoclonal antibody (Santa Cruz Biotechnology, Inc.), a rabbit anti-TLR8 polyclonal antibody (IMGENEX), or a rabbit anti-TLR2 polyclonal antibody (Rockland Immunochemicals) (1:100 dilution for all); and subsequently incubated with Texas RedR-X–conjugated goat anti-rabbit antibody (1:200 dilution; Invitrogen) for 1 h at RT or goat anti-mouse DyLight 594 (Thermo Scientific).

Techniques: Expressing, Infection, Incubation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phagosomal signaling by Borrelia burgdorferi in human monocytes involves Toll-like receptor (TLR) 2 and TLR8 cooperativity and TLR8-mediated induction of IFN-beta.

doi: 10.1073/pnas.1013776108

Figure Lengend Snippet: Fig. 3. Phagocytosed Bb colocalizes with TLR2 and TLR8 in human mono- cytes. A composite of representative confocal micrographs shows three consecutive Z-stack planes of GFP-Bb–stimulated human monocytes. Extra- cellular Bb interacts with TLR2 at the plasma membrane of the monocyte (A), whereas recently internalized (bacterial coil) and fully degraded (fluorescent bleb) Bb colocalizes with TLR2 (B) and TLR8 (C). Mander’s colocalization coefficient values (M2), shown in the upper right corner of each figure, are indicative of the proportion of the green signal overlapping with the signal in the red channel. Red depicts TLR2 signals in A and B and TLR8 in C, green depicts GFP-Bb, and white represents red and green pixels that colocalize. (Scale bar, 5 μm.) Colocalization through the Z-stack images illustrated in A and B (TLR2) and in C (TLR8) is shown in Movie S1, Movie S2, and Movie S3.

Article Snippet: Following different time points of stimulation (0 min, 30 min, 60 min, 90 min, 3 h, and 4 h), slides were fixed with 4% (vol/vol) paraformaldehyde for 10 min at room temperature (RT); permeabilized with 0.2% saponin for 10 min; blocked with Connaught Medical Research Laboratories (CMRL) 10% FCS for 2 h at RT; incubated overnight at 4 °C with a rabbit anti-human IRF7 polyclonal or mouse anti-human IRF3 monoclonal antibody (Santa Cruz Biotechnology, Inc.), a rabbit anti-TLR8 polyclonal antibody (IMGENEX), or a rabbit anti-TLR2 polyclonal antibody (Rockland Immunochemicals) (1:100 dilution for all); and subsequently incubated with Texas RedR-X–conjugated goat anti-rabbit antibody (1:200 dilution; Invitrogen) for 1 h at RT or goat anti-mouse DyLight 594 (Thermo Scientific).

Techniques: Clinical Proteomics, Membrane