Journal: iScience

Article Title: cGAS/STING sensing in dendritic cells discriminates between daptomycin sensitive and resistant Staphylococcus aureus clinical isolates

doi: 10.1016/j.isci.2026.115854

Figure Lengend Snippet: TLR2 is required for MutuDC sensing of, but not internalization of MRSA (A) Relative pHrodo labeled MRSA internalization by MutuDC over 4 h following stimulation with DapS A8819 (light blue symbols) or DapR A8817 (dark blue symbols), or media alone (white squares). Prior to stimulation, MutuDC were pre-treated for 1 h with TLR2 blocking antibody (clone T2.5; triangles with dashed lines) or media alone (circles with filled lines). Relative MRSA internalization by each DC subset is expressed as the gMFI of pHrodo. Results show the mean and (SD) of duplicates from one experiment, representative of two independent experiments. (B) Cytokine secretion (pg/mL) by MutuDC stimulated with TLR2 ligand peptidoglycan of S. aureus (PGN-SA) (10 μg/mL) or (C) DapS (A8819; light blue) or DapR (A8817; dark blue) MRSA (MOI of 10) for 18 h. MutuDC were first pre-treated with either TLR2 blocking antibody (dot-filled bars) or media alone (filled bars) as in A, or an isotype control (clone 163D3, empty bars) at 1 μg/mL. Results pooled from four (B) or three (C) independent experiments and expressed as the mean ± SEM, with each symbol (circle, square, and directional triangles) representing paired experimental replicates ( n = 3). Statistical significance determined using paired t test and reported as indicated by an ∗ when p ≤ 0.05. (D) Expression of surface activation markers by MutuDC stimulated with DapS A8819 MRSA. DC were pre-treated with TLR2 blocking antibody (black trace), isotype control (dashed red trace), and media alone (light blue shaded). Unstained control sample is shown for each marker (black dashed trace). Data shown from one experiment, representative of three independent experiments.

Article Snippet: mAB mTLR2- anti-mouse/human TLR2 , InvivoGen , Cat# mab-mtlr2; RRID: AB_763722.

Techniques: Labeling, Blocking Assay, Control, Expressing, Activation Assay, Marker

Journal: Brain, behavior, and immunity

Article Title: TLR2 immunotherapy suppresses neuroinflammation, tau spread, and memory loss in rTg4510 mice.

doi: 10.1016/j.bbi.2024.08.002

Figure Lengend Snippet: Fig. 1. TLR2 knockout reduces tau pathology and alleviates cognitive functions in rTg4510 mice. (A) Differential RNA expression of TLRs in AD brains compared to cognitively normal controls. Log2 fold changes are indicated. ACC, anterior cingulate cortex; CBE, cerebellum; DLPFC, dorsolateral prefrontal cortex; FP, frontal pole; IFG, inferior frontal gyrus; PCC, posterior cingulate cortex; PHG, parahippocampal gyrus; STG, superior temporal gyrus; TCX, temporal cortex. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (B-D) Tlr2 KO ameliorates cognitive impairment in rTg4510 mice. The 6-month-old mice (tTA/WT, tTA/Tlr2 KO, rTg4510/ WT, rTg4510/Tlr2 KO) were analyzed with the Y-maze test [B, two-way ANOVA with Tukey test, n (male, female) = 10 (7, 3); 10 (2, 8); 8 (6, 2); 11 (4, 7)], novel object recognition test [C, two-way ANOVA with Tukey test, n (male, female) = 8 (5, 3); 7 (2, 5); 6 (4, 2); 7 (2, 5)], and passive avoidance test [D, paired t-test, n (male, female) = 10 (7, 3); 10 (2, 8); 9 (6, 3); 11 (4, 7)]. (E-G) Tlr2 KO reduces tau pathology and microglial activation in rTg4510 mice. Immunohistochemistry of AT8 and Iba1 in the frontal cortex, dentate gyrus, and entorhinal cortex (E). Scale bar, 20 μm. Quantification of AT8 signal intensities (F) and the number of Iba1- positive cells (G). Two-way ANOVA with Tukey test, n = 7, 6, 3, 5. Data are represented as mean ± SEM. N.S., not significant.

Article Snippet: The primary antibodies used in the study: Tomaralimab (Amyloid Solution Inc., 1:250), anti-human TLR2 (Cell Signaling Technology #12276, 1:1000), anti-mouse TLR2 (Cell Signaling Technology #13744, 1:1000), anti-human tau HT7 (Invitrogen #MN1000, 1:1000), antiTau4R (Cell Signaling Technology #30328, 1:1000), anti-phospho-tau AT8 (Invitrogen #MN1020, 1:500), anti-Iba1 (GeneTex #GTX635400, 1:500), anti-Iba1 (Wako #019-19741, 1:1000), anti-MAP2 (Cell Signaling Technology #4542, 1:500), anti-NeuN (Millipore #MAB377, 1:1000), anti-GFAP (Cell Signaling Technology #80788, 1:1000), antiMyD88 (Cell Signaling Technology #50010, 1:1000), anti-NLRP3 (Cell Signaling Technology #15101, 1:1000), anti-β-actin (Santa Cruz Biotechnology #sc-47778, 1:1000), anti-GFP (Santa Cruz Biotechnology #sc-9996, 1:3000), anti-His-Tag (Cell Signaling Technology #2365, 1:1000).

Techniques: Knock-Out, RNA Expression, Activation Assay, Immunohistochemistry

Journal: Brain, behavior, and immunity

Article Title: TLR2 immunotherapy suppresses neuroinflammation, tau spread, and memory loss in rTg4510 mice.

doi: 10.1016/j.bbi.2024.08.002

Figure Lengend Snippet: Fig. 2. Microglial TLR2 binds to oligomeric tau. (A) Monomeric tau (mTau) and oligomeric tau (oTau) proteins prepared in vitro were subjected to native PAGE and stained with Coomassie Brilliant Blue or immunoblotted with anti-human tau (HT7) antibody. (B-C) TLR2 preferentially binds to oTau. Extracts of HEK293T cells transfected with TLR2-GFP were incubated without (None) or with either mTau or oTau (250 nM, 12 h) and subjected to immunoprecipitation (IP) (B). The signals of TLR2 on the blots were measured by ImageJ (C). One-way ANOVA with Holm-ˇSíd´ak test, n = 3. (D-E) Mapping of TLR2 domains involved in tau binding. A schematic diagram of TLR2 domains and mutants (ΔLBD, ΔDimer, D327W/F349L, P681H) (D). HEK293T cells were transfected with either WT or mutant TLR2-GFP. Cell lysates were incubated with 250 nM oTau and subjected to immunoprecipitation (IP) (E). (F-G) TLR2 activation increases binding of oligomeric tau. BV2 cells were left untreated (Nontreat) or incubated with Pam3CSK4 (10 μg/ml) overnight and then incubated with DyLight 488-labeled mTau or oTau (200 nM, 4 h). Cells were washed and immunostained with anti-TLR2 antibody (F). Scale bar, 10 μm. TLR2-bound DyLight 488 signals were measured by ImageJ (G). Two-way ANOVA with Tukey test, n = 3, 50 cells per group. Data are represented as mean ± SEM.

Article Snippet: The primary antibodies used in the study: Tomaralimab (Amyloid Solution Inc., 1:250), anti-human TLR2 (Cell Signaling Technology #12276, 1:1000), anti-mouse TLR2 (Cell Signaling Technology #13744, 1:1000), anti-human tau HT7 (Invitrogen #MN1000, 1:1000), antiTau4R (Cell Signaling Technology #30328, 1:1000), anti-phospho-tau AT8 (Invitrogen #MN1020, 1:500), anti-Iba1 (GeneTex #GTX635400, 1:500), anti-Iba1 (Wako #019-19741, 1:1000), anti-MAP2 (Cell Signaling Technology #4542, 1:500), anti-NeuN (Millipore #MAB377, 1:1000), anti-GFAP (Cell Signaling Technology #80788, 1:1000), antiMyD88 (Cell Signaling Technology #50010, 1:1000), anti-NLRP3 (Cell Signaling Technology #15101, 1:1000), anti-β-actin (Santa Cruz Biotechnology #sc-47778, 1:1000), anti-GFP (Santa Cruz Biotechnology #sc-9996, 1:3000), anti-His-Tag (Cell Signaling Technology #2365, 1:1000).

Techniques: In Vitro, Clear Native PAGE, Staining, Transfection, Incubation, Immunoprecipitation, Binding Assay, Mutagenesis, Activation Assay, Labeling

Journal: Brain, behavior, and immunity

Article Title: TLR2 immunotherapy suppresses neuroinflammation, tau spread, and memory loss in rTg4510 mice.

doi: 10.1016/j.bbi.2024.08.002

Figure Lengend Snippet: Fig. 4. Tau-induced TLR2 activation in microglia promotes neuronal tau uptake. (A-B) Microglial TLR2 increases neuronal tau uptake in neuron-microglia co- culture system. WT and Tlr2 KO mouse primary cortical neurons (DIV 7) and microglia (DIV 14) were co-cultured and treated with DyLight 488-oligomeric tau (200 nM, 24 h) (A). Scale bar, 10 μm. DyLight 488 intensities in MAP2-positive neurons (arrowheads) were measured (B). Two-way ANOVA with Tukey test, n = 3, 28–58 cells per group. (C-D) Tlr2 deficiency reduces tau-induced microglial activation in the mouse hippocampus. Tau oligomers (6 μg) were intracranially injected into the hippocampus of 4-month-old mice. After 7 days, brain sections were immunostained with anti-human tau (HT7) and anti-Iba1 antibodies (C). Scale bar, 50 μm. Manders’ colocalization coefficient (fraction of HT7 overlapping Iba1) was measured (D). Unpaired t-test, two-tailed, n = 3 per group. (E-F) Tlr2 deficiency reduces neuronal tau uptake in the mouse hippocampus. Tau oligomers (6 μg) were intracranially injected into the hippocampus of 3-month-old mice. After 48 h, brain sections were immunostained with anti-human tau (HT7) and anti-MAP2 antibodies (E). Arrowheads indicate internalized tau oligomers. Scale bar, 10 μm. Per- centages of HT7-positive neurons in the injection area were estimated (F). Unpaired t-test, two-tailed, n = 3 per group. Data are represented as mean ± SEM.

Article Snippet: The primary antibodies used in the study: Tomaralimab (Amyloid Solution Inc., 1:250), anti-human TLR2 (Cell Signaling Technology #12276, 1:1000), anti-mouse TLR2 (Cell Signaling Technology #13744, 1:1000), anti-human tau HT7 (Invitrogen #MN1000, 1:1000), antiTau4R (Cell Signaling Technology #30328, 1:1000), anti-phospho-tau AT8 (Invitrogen #MN1020, 1:500), anti-Iba1 (GeneTex #GTX635400, 1:500), anti-Iba1 (Wako #019-19741, 1:1000), anti-MAP2 (Cell Signaling Technology #4542, 1:500), anti-NeuN (Millipore #MAB377, 1:1000), anti-GFAP (Cell Signaling Technology #80788, 1:1000), antiMyD88 (Cell Signaling Technology #50010, 1:1000), anti-NLRP3 (Cell Signaling Technology #15101, 1:1000), anti-β-actin (Santa Cruz Biotechnology #sc-47778, 1:1000), anti-GFP (Santa Cruz Biotechnology #sc-9996, 1:3000), anti-His-Tag (Cell Signaling Technology #2365, 1:1000).

Techniques: Activation Assay, Co-Culture Assay, Cell Culture, Injection, Two Tailed Test

Journal: PLoS ONE

Article Title: miR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells

doi: 10.1371/journal.pone.0068296

Figure Lengend Snippet: ( A ) TLR2 expression levels were analyzed by real-time PCR following transfection with 25 nM miR-1225-5p mimic. Increasing the amount of miR-1225-5p within ARPCs resulted in a 1.3 fold reduction of TLR2 mRNA levels 24 hours after transfection. Expression data were normalized on the housekeeping gene β-actin. Data are representative of three independent experiments (means ± SEM), *p<0.01. ( B–C ) TLR2 protein expression after transfection with 50 nM miR-1225-5p mimic. A strong reduction of TLR2 in ARPC was found after 3 days from transfection with miR-1225-5p mimic, as shown by immunofluorescence staining. To-pro-3 counterstains nuclei (blue). Original view X63.

Article Snippet: The cells were blocked for 1 h (BSA in PBS, pH 7.4) and then incubated with a mouse anti-human TLR2 mAb (Hycult Biotechnology) and a mouse anti-human CD133 mAb (Miltenyi Biotec) overnight at 4°C.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Immunofluorescence, Staining