Journal: Journal of Leukocyte Biology

Article Title: CD68 on rat macrophages binds tightly to S100A8 and S100A9 and helps to regulate the cells’ immune functions

doi: 10.1189/jlb.2a0415-170rrr

Figure Lengend Snippet: Figure 8. Inhibitory effects of specific antibodies for CD68, TLR4, and RAGE on the expression of mRNAs for S100A8 and S100A9 in macrophages. We measured the mRNA expression levels of S100A8 and S100A9 to investigate the effects of specific antibodies for CD68, TLR4, and RAGE on the immune functions of macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) using a StepOnePlus Real-Time PCR System. (A) RT-PCR was carried out to detect S100A8 mRNA in macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) for 0.5 h in 5% CO2 at 37°C in the presence and absence of each antibody. (B) RT-PCR was also carried out to detect S100A9 mRNA in macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) alone for 0.5 h in 5% CO2 at 37°C in the presence and absence of each antibody. Error bars represent the SD (n = 3) of independent experiments. In this study, the experimental procedures have been repeated 3 times. Statistical analysis was performed using Dunnett’s test; *P , 0.05.

Article Snippet: Anti-TLR4 mAb was obtained from Novus Biologicals (Littleton, CO, USA; NB100-56566).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Leukocyte Biology

Article Title: CD68 on rat macrophages binds tightly to S100A8 and S100A9 and helps to regulate the cells’ immune functions

doi: 10.1189/jlb.2a0415-170rrr

Figure Lengend Snippet: Figure 9. Inhibitory effects of specific antibodies for CD68, TLR4, and RAGE on the expression of mRNAs for inflammatory and anti-inflammatory cytokines in macrophages. We measured the mRNA expression levels of IL-1b, TNF-a, IL-6, IL- 10, and TGF-b to investigate the effects of specific antibodies for CD68, TLR4, and RAGE on the immune functions of macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) alone using a StepOnePlus Real-Time PCR System. (A–E) RT-PCR was carried out to detect these cytokine mRNAs in macrophages. Changes in the mRNA expression levels of IL-1b (Α), TNF-a (Β), IL-6 (C), IL-10 (D), and TGF-b (E), respectively, in macrophages that had been activated with r-S100A8 (10 mg/ml) or r-S100A9 (10 mg/ml) alone for 0.5 h in 5% CO2 at 37°C in the presence and absence of each antibody. Error bars represent the SD (n = 3) of independent experiments. In this study, the experimental procedures have been repeated 3 times. Statisti- cal analysis was performed using Dunnett’s test; *P , 0.05.

Article Snippet: Anti-TLR4 mAb was obtained from Novus Biologicals (Littleton, CO, USA; NB100-56566).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in cell and developmental biology

Article Title: Engineered Extracellular Vesicles Loaded With miR-124 Attenuate Cocaine-Mediated Activation of Microglia.

doi: 10.3389/fcell.2020.00573

Figure Lengend Snippet: FIGURE 2 | Dendritic cell-derived EVs loaded with Cy5-miR-124 are taken up by microglia and inhibit cocaine-mediated microglial activation. (A) Representative images of mouse primary microglia exposed to EVs transfected with Cy5-miR-124 or Cy5-scrambled miRNA (1 h) showing the presence of intracellular Cy5. (B–E) Expression of TLR4, STAT3, ERK and Iba-1 in mouse primary microglial cells pretreated with EVs loaded with miR-124 mimic or scrambled miRNA followed by exposure to cocaine (10 µM). All experiments were done at least three independent times, and representative figures are shown. Actin served as a loading control for western blots. Quantification of western blots is shown under each blot. Data are shown as mean ± SEM. ∗p < 0.05 vs. control, #p < 0.05 vs. cocaine group.

Article Snippet: Antibodies and reagents used in this work were purchased from the indicated sources: Lamp2 (NB300-591; Novus, Centennial, CO, United States); CD63 (ab216130; Abcam, Cambridge, MA, United States), TLR4 (NB100-56566 Novus, Centennial, CO, United States), Iba1 (Novus NB1001028 or Wako; 19-19741), STAT3 (4904S, Cell Signaling, MA, United States), ERK1 /2 (9107S; Cell Signaling, MA, United States), goat anti-mouseHRP (Santa Cruz Biotechnology; sc-2005), and goat anti-rabbitHRP (Santa Cruz Biotechnology; sc-2004); NFkB p65 (16502, Abcam, Cambridge, MA, United States); MYD88 (ab2064; Abcam, Cambridge, MA, United States); β-Actin (A1978; SigmaAldrich, St. Louis, MO, United States).

Techniques: Derivative Assay, Activation Assay, Transfection, Expressing, Control, Western Blot

Journal: Frontiers in cell and developmental biology

Article Title: Engineered Extracellular Vesicles Loaded With miR-124 Attenuate Cocaine-Mediated Activation of Microglia.

doi: 10.3389/fcell.2020.00573

Figure Lengend Snippet: FIGURE 4 | Intranasal delivery of dendritic cell-derived EVs loaded with Cy5-miR-124 inhibits cocaine-mediated upregulation of TLR4, MYD88, STAT3 and NF-kB p65 in vivo. Wild-type C57BL/6N, mice were administered either cocaine (20 mg/kg; i.p.) or saline for seven consecutive days. On the fifth, sixth, and seventh days, mice were intranasally administered dendritic cell-derived EVs loaded with Cy5-miR-124 or Cy5-scrambled miRNAs or were left unloaded, followed by cocaine injection. (A–D) Western blot images depicting the effect of EV-miR-124 on the expression of TLR4, MYD88, STAT3 and NF-kB p65. Actin served as a loading control. Quantification of western blots is shown under each blot. Data are shown as mean ± SEM (n = 4/group, ∗p < 0.05 vs. saline+ EV control, #p < 0.05 vs. cocaine+ EV control group).

Article Snippet: Antibodies and reagents used in this work were purchased from the indicated sources: Lamp2 (NB300-591; Novus, Centennial, CO, United States); CD63 (ab216130; Abcam, Cambridge, MA, United States), TLR4 (NB100-56566 Novus, Centennial, CO, United States), Iba1 (Novus NB1001028 or Wako; 19-19741), STAT3 (4904S, Cell Signaling, MA, United States), ERK1 /2 (9107S; Cell Signaling, MA, United States), goat anti-mouseHRP (Santa Cruz Biotechnology; sc-2005), and goat anti-rabbitHRP (Santa Cruz Biotechnology; sc-2004); NFkB p65 (16502, Abcam, Cambridge, MA, United States); MYD88 (ab2064; Abcam, Cambridge, MA, United States); β-Actin (A1978; SigmaAldrich, St. Louis, MO, United States).

Techniques: Derivative Assay, In Vivo, Saline, Injection, Western Blot, Expressing, Control

Journal: Journal of neuroinflammation

Article Title: Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

doi: 10.1186/s12974-023-02712-1

Figure Lengend Snippet: Fig. 1 Cre-mediated recombination selectively depletes TLR4 on endothelial cells. Representative retinal whole mounts of TekCretdTomato mice stained with Iba-1 (green) in naïve mice (A–C) and LPS-challenged mice (E–G). tdTomato (magenta) was selectively expressed in retinal blood vessels and no co-localization with Iba-1 was detected. The yellow squares in C and G are magnified in D and H, respectively. Scale bars: 100 μm. Representative dot plots of single, live, TIE2posTLR4pos cells in the retinas of naïve TekCre−negTlr4loxP/loxP and TekCre−posTlr4loxP/loxP mice (I, J). The majority of TIE2pos cells (72.7%) in the TekCre−negTlr4loxP/loxP retinas express TLR4, while in the retinas of TekCre−posTlr4loxP/loxP mice only 6.4% of TIE2pos cells are TLR4pos (K; n = 3 mice per group). The data were analyzed with 2-tailed unpaired t-test, ***p < 0.001)

Article Snippet: Single-cell suspensions were stained with a TIE2 antibody (TEK4; 1:100; allophycocyanin/APC; Cat # 124009) and a primary mouse monoclonal TLR4 antibody (76B357.1; 1:400; Novus Biologicals, Littleton, CO, USA; Cat # NB100-56566SS), followed by incubation with a secondary goat F(ab′)2 anti-mouse IgG H&L (phycoerythrin, PE) pre-adsorbed antibody (1:100; Cat # ab7002; Abcam, Cambridge, United Kingdom).

Techniques: Staining

Journal: Journal of neuroinflammation

Article Title: Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

doi: 10.1186/s12974-023-02712-1

Figure Lengend Snippet: Fig. 2 Systemic LPS fails to trigger microglia activation and retinal vasodilation in endothelial Tlr4-deficient mice. Representative images of Iba-1-stained retinal whole mounts in naïve (A, G, M) and LPS-challenged (D, J, P) C57BL/6J, TekCre−negTlr4loxP/loxP and TekCre−posTlr4loxP/loxP mice, respectively. Iba-1pos cells located in the GCL-INL and the OPL are shown in magenta and white, respectively. Yellow arrows in D indicate the clustering of Iba-1pos cells around a retinal vessel. In naïve C57BL/6J mice, microglia cells have a ramified morphology throughout the retina (B, C). After the LPS challenge, Iba-1pos cells adopt a bushy or amoebic morphology and accumulate around retinal blood vessels in the GCL-INL (E). In the OPL, these cells have an amoebic or rod-like shape (F). The morphology of Iba-1pos cells before or after the LPS challenge in TekCre−negTlr4loxP/loxP mice was comparable to C57BL/6J mice (H, I, K, L). Individual Iba-1pos cells of TekCre−posTlr4loxP/loxP mice had a ramified morphology both in the naïve and the LPS-challenged group (N, O, Q, R). Quantification of average branch length (S; n = 4–5 mice per group), number of endpoints per cell (T; n = 4–5 mice per group) and Iba-1 occupied volume (U; n = 4–5 mice per group) on images obtained from retinal whole mounts. Reduced branch length, less endpoints per cell and increased Iba-1 occupied volume were detected in the C57BL/6J but not the endothelial Tlr4 knockout mice. Representative images of fluorescein angiographs at baseline (V, X) and after the LPS challenge (W, Y) in C57BL/6J and TekCre−posTlr4loxP/loxP mice, respectively and quantification of vein dilation (Z; n = 5–7 mice per group). Vein diameter was significantly increased in LPS-challenged C57BL/6J mice compared to baseline, but not in TekCre−posTlr4loxP/loxP mice. The quantification data are reported as mean ± SD. The data were analyzed separately for each genotype with 2-tailed unpaired t-test (S–U) or 2-tailed paired t-test (Z). **p < 0.01, ***p < 0.001. a Artery; v vein; GCL-INL ganglion cell layer-inner nuclear layer; OPL outer plexiform layer. Scale bars: left panel: 100 μm; middle and right panels: 50 μm

Article Snippet: Single-cell suspensions were stained with a TIE2 antibody (TEK4; 1:100; allophycocyanin/APC; Cat # 124009) and a primary mouse monoclonal TLR4 antibody (76B357.1; 1:400; Novus Biologicals, Littleton, CO, USA; Cat # NB100-56566SS), followed by incubation with a secondary goat F(ab′)2 anti-mouse IgG H&L (phycoerythrin, PE) pre-adsorbed antibody (1:100; Cat # ab7002; Abcam, Cambridge, United Kingdom).

Techniques: Activation Assay, Staining, Knock-Out

Journal: Journal of neuroinflammation

Article Title: Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

doi: 10.1186/s12974-023-02712-1

Figure Lengend Snippet: Fig. 3 Effect of LPS on microglia/macrophage numbers in the presence or absence of endothelial Tlr4. Representative dot plots of CD11bpos populations (A, B left panels) gated as CD11bposCD45low/neg microglia and CD11bposCD45hi monocyte-derived macrophages (A, B right panels) in naïve and LPS-challenged C57BL/6J and TekCre−posTlr4loxP/loxP mice, respectively. Quantification of CD45low/neg microglia and CD45hi macrophages expressed as a percentage of the total number of live cells in the samples (C, D) revealed an increase of both populations after the LPS challenge in C57BL/6J mice but not in TekCre−posTlr4loxP/loxP mice (n = 5 mice per genotype and experimental group). The data were analyzed separately for each genotype with 2-tailed unpaired t-test (***p < 0.001). Representative images of retinal sections stained with Iba-1 and ICAM-1 in naïve (E, G, I) and LPS-challenged (F, H, J) C57BL/6J, TekCre−negTlr4loxP/loxP and TekCre−posTlr4loxP/loxP mice, respectively. ICAM-1 immunoreactivity was detected in LPS-challenged C57BL/6J and TekCre−negTlr4loxP/loxP mice but not in TekCre−posTlr4loxP/loxP mice. Scale bars: 200 μm. GCL Ganglion cells layer; INL inner nuclear layer; IPL inner plexiform layer; ONL outer nuclear layer; OPL outer plexiform layer

Article Snippet: Single-cell suspensions were stained with a TIE2 antibody (TEK4; 1:100; allophycocyanin/APC; Cat # 124009) and a primary mouse monoclonal TLR4 antibody (76B357.1; 1:400; Novus Biologicals, Littleton, CO, USA; Cat # NB100-56566SS), followed by incubation with a secondary goat F(ab′)2 anti-mouse IgG H&L (phycoerythrin, PE) pre-adsorbed antibody (1:100; Cat # ab7002; Abcam, Cambridge, United Kingdom).

Techniques: Derivative Assay, Staining

Journal: Journal of neuroinflammation

Article Title: Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

doi: 10.1186/s12974-023-02712-1

Figure Lengend Snippet: Fig. 4 Endothelial Tlr4 depletion rescue the retina from LPS-induced visual dysfunction. Representative waveforms at baseline (A, C, E) and after the LPS challenge (B, D, F) under scotopic conditions in C57BL/6J, TekCre−negTlr4loxP/loxP and TekCre−posTlr4loxP/loxP mice, respectively. Comparison of a- (G, I, K) and b-wave amplitudes (H, J, L) between baseline and LPS in C57BL/6J, TekCre−negTlr4loxP/loxP and TekCre−posTlr4loxP/loxP mice, respectively. Reduced mean a- and b-wave amplitudes were recorded in C57BL/6J and TekCre−negTlr4loxP/loxP mice after the LPS challenge. In TekCre−posTlr4loxP/loxP mice, mean a- and b-wave amplitudes after the LPS challenge were comparable to baseline levels. The data were analyzed with repeated measures 2-way ANOVA followed by Sidak’s post hoc analysis (n = 4–5 mice per genotype, one eye per mouse; *p < 0.05, **p < 0.01, ***p < 0.001)

Article Snippet: Single-cell suspensions were stained with a TIE2 antibody (TEK4; 1:100; allophycocyanin/APC; Cat # 124009) and a primary mouse monoclonal TLR4 antibody (76B357.1; 1:400; Novus Biologicals, Littleton, CO, USA; Cat # NB100-56566SS), followed by incubation with a secondary goat F(ab′)2 anti-mouse IgG H&L (phycoerythrin, PE) pre-adsorbed antibody (1:100; Cat # ab7002; Abcam, Cambridge, United Kingdom).

Techniques: Comparison

Journal: Journal of neuroinflammation

Article Title: Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

doi: 10.1186/s12974-023-02712-1

Figure Lengend Snippet: Fig. 6 Proposed mechanism of systemic LPS-induced retinal microglia activation and retinal dysfunction. A. The inner BRB (iBRB) is composed by a deep, an intermediate, and a superficial vascular plexus. In the healthy retina, endothelial cells, that line the blood vessels, are connected by tight junctions forming an effective vascular barrier. Endothelial cells bear TLR4 receptors and they are surrounded by pericytes and perivascular macrophages. Microglia resides in close proximity to the vasculature, while monocytes circulate in the bloodstream. B. Upon the systemic LPS challenge, LPS binds to TLR4 located on circulating monocytes and endothelial cells (1), and induces the expression of the adhesion molecule ICAM-1 from the latter (2). Activated endothelial cells release cytokines and chemokines (3), which may act as chemoattractants causing microglia migration towards the affected vasculature (4). Subsequently, monocyte-derived macrophages are entering the retina through the disrupted BRB and AIF-1 – ICAM-1 interactions (5). Migration of microglia away from retinal neurons may account for disruption of ribbon synapses and impaired retinal function (6). EC endothelial cell; GCL ganglion cell layer; iBRB inner blood retinal barrier; ICAM-1 intercellular molecule 1; INL inner nuclear layer; IPL inner plexiform layer; LFA-1 lymphocyte function-associated antigen 1; LPS lipopolysaccharide; M microglia; MDMs monocyte-derived macrophages; ONL outer nuclear layer; OPL outer plexiform layer; PRs photoreceptors; PVMs perivascular macrophages; RSs ribbon synapses; TJs tight junctions; TLR4 Toll-like receptor 4

Article Snippet: Single-cell suspensions were stained with a TIE2 antibody (TEK4; 1:100; allophycocyanin/APC; Cat # 124009) and a primary mouse monoclonal TLR4 antibody (76B357.1; 1:400; Novus Biologicals, Littleton, CO, USA; Cat # NB100-56566SS), followed by incubation with a secondary goat F(ab′)2 anti-mouse IgG H&L (phycoerythrin, PE) pre-adsorbed antibody (1:100; Cat # ab7002; Abcam, Cambridge, United Kingdom).

Techniques: Activation Assay, Expressing, Migration, Derivative Assay, Disruption

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Toll-like receptor 4 activation attenuates profibrotic response in control lung fibroblasts but not in fibroblasts from patients with IPF.

doi: 10.1152/ajplung.00119.2016

Figure Lengend Snippet: Fig. 1. Toll-like receptor 4 (TLR4) is present in fibroblast foci (FF) in idiopathic pulmonary fibrosis (IPF) lungs. A: representative immunofluorescence images of IPF lung sections (30 m) acquired by laser-scanning microscope (LSM). FF were stained with DAPI (blue, nuclei), -smooth muscle actin (-SMA) (red, myofibroblasts), and TLR4 (green). A’ shows merged image with xz-projections (bottom) obtained from 3-dimensional stacks of consecutive optical sections. Scale bar 15 m. B: hematoxylin and eosin (H&E) staining of FF (arrows) in patient with IPF. C: corresponding immunohistochemistry staining for TLR4, positive in fibroblasts of FF. 1 representative patient out of 5 is shown. Scale bar 40 m. D: laser capture microdissection with subsequent RT-qPCR analysis shows expression of TLR4 in FF (n 5 individual patients) and surrounding tissue (n 3 individual patients) normalized to the control gene 2M.

Article Snippet: Sections were incubated with mouse monoclonal antibody against TLR4 (Novus Biologicals, Littleton, CO).

Techniques: Laser-Scanning Microscopy, Staining, Immunohistochemistry, Laser Capture Microdissection, Quantitative RT-PCR, Expressing, Control

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Toll-like receptor 4 activation attenuates profibrotic response in control lung fibroblasts but not in fibroblasts from patients with IPF.

doi: 10.1152/ajplung.00119.2016

Figure Lengend Snippet: Fig. 2. TLR4 is expressed on fibroblasts isolated from controls and IPF lungs. A: fibroblasts were characterized by staining for the myofibroblast marker -SMA (green, top) and for fibroblast marker TE-7 (red, bottom) in control (normal human lung fibroblasts, NL-FB, left) and IPF fibroblasts (right). Scale bar 40 m. B: TLR4 gene expressions assessed by RT-qPCR at baseline show similar expression levels in control (NL-FB, n 3 individual patients), IPF (n 3 individual patients), and normal human lung fibroblasts (NHLF, n 3 independent experiments). Data of each patient/experiment in triplicate were normalized to 2M and expressed as means ( SE). C: TLR4 (100 kDa) protein expression of whole cell lysates assessed by Western blotting in control (NL-FB, n 4 individual patients) and IPF fibroblasts (n 3 individual patients). A549 epithelial cell line lysates served as positive control. D: quantification of TLR4 protein expression by normalization to the loading control -actin (45 kDa) confirmed similar TLR4 levels in control (NL-FB) and IPF fibroblasts. Data are expressed as means ( SE).

Article Snippet: Sections were incubated with mouse monoclonal antibody against TLR4 (Novus Biologicals, Littleton, CO).

Techniques: Isolation, Staining, Marker, Control, Quantitative RT-PCR, Expressing, Western Blot, Positive Control

Journal: Frontiers in Pharmacology

Article Title: Berberine Attenuates Intestinal Mucosal Barrier Dysfunction in Type 2 Diabetic Rats

doi: 10.3389/fphar.2017.00042

Figure Lengend Snippet: The gut-protective effects of BBR are related to TLR4/MyD88/NFκB signaling pathway. (A) TLR4 expressions in intestine ( n = 4). (B) MyD88 expressions in intestine ( n = 3). (C) Fold change of p-IKKβ/IKKβ ratios in intestine ( n = 4). (D) The mRNA levels of CD14 ( n = 6). (E) The mRNA levels of LBP ( n = 6). The levels of mRNA were expressed relative to the β-actin. The protein sampling mass was 200 μg for TLR4 and MyD88; while 100 μg for p-IKKβ and IKKβ. Data presented as mean ± SD. ∗ P < 0.05 vs. NO group, ∗∗ P < 0.01 vs. NO group, # P < 0.05 vs. MO group, ## P < 0.01 vs. MO group.

Article Snippet: Proteins of intestines (different sampling volumes for various antibodies) were separated through gel electrophoresis, transferred onto NC membranes, and incubated with antibodies against TLR4 (NOVUS, USA), Occludin-1 (OCLN, Gene Tex, USA), ZO-1 (Invitrogen, USA), Claudin-1 (Abcam, UK), MyD88 (CST, USA), p-IKKβ (Gene Tex, USA),IKKβ (Gene Tex, USA) and β-actin (Affinity, USA).

Techniques: Sampling