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rabbit anti mouse tlr2 antibody (Bioss)

Name: rabbit anti mouse tlr2 antibody
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Rating: 95 (112 reviews)

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Bioss rabbit anti mouse tlr2 antibody
Rabbit Anti Mouse Tlr2 Antibody, supplied by Bioss, used in various techniques. Bioz Stars score: 95/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 95 stars, based on 112 article reviews

rabbit anti mouse tlr2 antibody - by Bioz Stars, 2026-02

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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.

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

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.

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.

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

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

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.

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

OAA suppressed infiltration of immune cells and macrophages in the spinal cords of EAE mice. H&E staining showed that OAA administration attenuated inflammatory infiltration in spinal cords compared to the infiltration observed in EAE mice (upper panel). Immunohistochemistry showed that infiltration of CD68+ macrophages was reduced in the spinal cords of OAA-treated mice (lower panel). Expression of TLR2 was elevated in EAE spinal cords and mainly expressed in CD68 + macrophages (lower panel). Scale bars = 100 µm. OAA, oleanolic acid acetate; EAE, experimental autoimmune encephalomyelitis; H&E, hematoxylin and eosin.

Journal: Frontiers in Pharmacology

Article Title: Oleanolic Acid Acetate Alleviates Symptoms of Experimental Autoimmune Encephalomyelitis in Mice by Regulating Toll-Like Receptor 2 Signaling

doi: 10.3389/fphar.2020.556391

Figure Lengend Snippet: OAA suppressed infiltration of immune cells and macrophages in the spinal cords of EAE mice. H&E staining showed that OAA administration attenuated inflammatory infiltration in spinal cords compared to the infiltration observed in EAE mice (upper panel). Immunohistochemistry showed that infiltration of CD68+ macrophages was reduced in the spinal cords of OAA-treated mice (lower panel). Expression of TLR2 was elevated in EAE spinal cords and mainly expressed in CD68 + macrophages (lower panel). Scale bars = 100 µm. OAA, oleanolic acid acetate; EAE, experimental autoimmune encephalomyelitis; H&E, hematoxylin and eosin.

Article Snippet: The slides were incubated overnight with rabbit polyclonal anti-mouse TLR2 (1:100, Thermo Fisher Scientific Inc., Waltham, MA, USA), and rat monoclonal anti-mouse CD68 (1:100, Thermo Fisher Scientific Inc., Waltham, MA, USA) antibodies at 4°C.

Techniques: Staining, Immunohistochemistry, Expressing

OAA downregulated the expression of TLR2 and downstream signaling molecules in the spinal cords of EAE mice. OAA downregulated TLR2 and MyD88 mRNA expression in the spinal cords (A) . Western blot analysis of TLR2, MyD88, IRAK4, and TRAF6 (B) . The expression level of TLR2, MyD88, IRAK4, and TRAF6 was significantly suppressed in OAA treated group, compared with that of EAE group (B) . Data are shown as mean ± standard error (SE), n=6, *p < 0.05 compared to EAE mice. OAA, oleanolic acid acetate; EAE, experimental autoimmune encephalomyelitis; TLR2, Toll-like receptor 2; MyD88, myeloid differentiation primary response protein 88; IRAK4, IL-1 receptor-associated kinase 4: TRAF6, tumor necrosis factor (TNF) receptor-associated factor 6.

Journal: Frontiers in Pharmacology

Article Title: Oleanolic Acid Acetate Alleviates Symptoms of Experimental Autoimmune Encephalomyelitis in Mice by Regulating Toll-Like Receptor 2 Signaling

doi: 10.3389/fphar.2020.556391

Figure Lengend Snippet: OAA downregulated the expression of TLR2 and downstream signaling molecules in the spinal cords of EAE mice. OAA downregulated TLR2 and MyD88 mRNA expression in the spinal cords (A) . Western blot analysis of TLR2, MyD88, IRAK4, and TRAF6 (B) . The expression level of TLR2, MyD88, IRAK4, and TRAF6 was significantly suppressed in OAA treated group, compared with that of EAE group (B) . Data are shown as mean ± standard error (SE), n=6, *p < 0.05 compared to EAE mice. OAA, oleanolic acid acetate; EAE, experimental autoimmune encephalomyelitis; TLR2, Toll-like receptor 2; MyD88, myeloid differentiation primary response protein 88; IRAK4, IL-1 receptor-associated kinase 4: TRAF6, tumor necrosis factor (TNF) receptor-associated factor 6.

Techniques: Expressing, Western Blot

The TLR-stimulant content of some processed foods can greatly exceed that of the normal mouse ileal microbiota. (A–C) Soluble TLR2, TLR4, and TLR5-stimulants were quantified in processed foods ( n = 14), mouse chow ( n = 6), mouse ileal ingesta ( n = 5), mouse feces ( n = 10) and human feces ( n = 6) using HEK-293-TLR transfectants. (D-F) Pro-inflammatory cytokine production by primary human monocytes ( n = 3 donors) exposed to extracts of mouse ileal ingesta, minced meat or chocolate ( n = 5 extracts of each type), diluted 1:10 (wt/vol) in culture medium. P -values vs. ileal contents, ANOVA with Tukey's test.

Journal: Frontiers in Immunology

Article Title: Dietary Toll-Like Receptor Stimulants Promote Hepatic Inflammation and Impair Reverse Cholesterol Transport in Mice via Macrophage-Dependent Interleukin-1 Production

doi: 10.3389/fimmu.2019.01404

Figure Lengend Snippet: The TLR-stimulant content of some processed foods can greatly exceed that of the normal mouse ileal microbiota. (A–C) Soluble TLR2, TLR4, and TLR5-stimulants were quantified in processed foods ( n = 14), mouse chow ( n = 6), mouse ileal ingesta ( n = 5), mouse feces ( n = 10) and human feces ( n = 6) using HEK-293-TLR transfectants. (D-F) Pro-inflammatory cytokine production by primary human monocytes ( n = 3 donors) exposed to extracts of mouse ileal ingesta, minced meat or chocolate ( n = 5 extracts of each type), diluted 1:10 (wt/vol) in culture medium. P -values vs. ileal contents, ANOVA with Tukey's test.

Article Snippet: Rabbit anti-mouse TLR2 (Santa-cruz, sc10739, 1:100) and rabbit anti-mouse TLR4 (Santa-cruz, sc10741, 1:100) used AF594-labeled goat anti-rabbit secondary (Invitrogen, A-11037, 1:500).

Techniques: Chocolate

The accumulation of TLR-stimulants in processed meat is dependent on the food microbiota. (A) Human primary monocytes were treated with filter-sterilized extracts of minced meat or diced onion, 1 ng/ml E. coli LPS or 100 ng/ml Pam 3 CSK 4 , with or without oxidized palmitoyl arachidonly phosphatidyl choline (OxPAPC, an inhibitor of TLR2 and TLR4, n = 4 volunteers). (B) NF-κB activation in HEK-293-TLR2 transfectants challenged with filter-sterilized extracts of minced meat samples when fresh, or at the ‘best before date’ (d7), with or without treatment with the antibiotics penicillin and streptomycin (P/S), or polymyxin-B (PMB, which specifically targets Gram-negative bacteria), or autoclaving before storage. (C) Cytokine responses of human primary monocytes to the same meat extracts ( n = 4 per group). (D) Total 16S rDNA in stool and food samples measured by qPCR (a culture-independent measure of historical bacterial activity). (E) Proportion of proteobacterial 16S rDNA in human and mouse fecal samples and meat and chocolate samples ( n = 5–7). (F) Relative biological activities of soluble TLR2- and TLR4-stimulants in filter-sterilized conditioned media of representative proteobacterial and non-proteobacterial organisms of the food and commensal microbiota (grown to A600 = 1.0), measured using HEK-293-TLR transfectants calibrated with Pam 3 CSK 4 and E. coli LPS, respectively. (G) Inflammatory cytokine production by primary human monocytes cultured with 1:1,000 dilutions of the same conditioned media. (H) NF-κB reporter responses in HEK-293 cells transfected with NOD-1 and filter sterilized homogenates of minced meats. Error bars shown are SEM. P -values vs. control condition (medium alone), ANOVA with Dunnett's test.

Journal: Frontiers in Immunology

Article Title: Dietary Toll-Like Receptor Stimulants Promote Hepatic Inflammation and Impair Reverse Cholesterol Transport in Mice via Macrophage-Dependent Interleukin-1 Production

doi: 10.3389/fimmu.2019.01404

Figure Lengend Snippet: The accumulation of TLR-stimulants in processed meat is dependent on the food microbiota. (A) Human primary monocytes were treated with filter-sterilized extracts of minced meat or diced onion, 1 ng/ml E. coli LPS or 100 ng/ml Pam 3 CSK 4 , with or without oxidized palmitoyl arachidonly phosphatidyl choline (OxPAPC, an inhibitor of TLR2 and TLR4, n = 4 volunteers). (B) NF-κB activation in HEK-293-TLR2 transfectants challenged with filter-sterilized extracts of minced meat samples when fresh, or at the ‘best before date’ (d7), with or without treatment with the antibiotics penicillin and streptomycin (P/S), or polymyxin-B (PMB, which specifically targets Gram-negative bacteria), or autoclaving before storage. (C) Cytokine responses of human primary monocytes to the same meat extracts ( n = 4 per group). (D) Total 16S rDNA in stool and food samples measured by qPCR (a culture-independent measure of historical bacterial activity). (E) Proportion of proteobacterial 16S rDNA in human and mouse fecal samples and meat and chocolate samples ( n = 5–7). (F) Relative biological activities of soluble TLR2- and TLR4-stimulants in filter-sterilized conditioned media of representative proteobacterial and non-proteobacterial organisms of the food and commensal microbiota (grown to A600 = 1.0), measured using HEK-293-TLR transfectants calibrated with Pam 3 CSK 4 and E. coli LPS, respectively. (G) Inflammatory cytokine production by primary human monocytes cultured with 1:1,000 dilutions of the same conditioned media. (H) NF-κB reporter responses in HEK-293 cells transfected with NOD-1 and filter sterilized homogenates of minced meats. Error bars shown are SEM. P -values vs. control condition (medium alone), ANOVA with Dunnett's test.

Techniques: Activation Assay, Bacteria, Activity Assay, Chocolate, Cell Culture, Transfection, Control

Development of an acute murine model of oral-LPS induced APR and dyslipidaemia. Three methods previously reported to increase the sensitivity of mice to circulating LPS were tested, (i) intraperitoneal administration of heat-killed Propionibacterium acnes ( n = 6 per group), (ii) 4 weeks priming with high fat diet (HFD, n = 10 per group) or (iii) 4 weeks priming with high cholesterol diet (HCD), ( n = 12 per group) ( , , ) (A) Hepatic F4/80 +ve (green) cell number was significantly elevated by all three treatments relative to control mice fed normal chow. (B) HCD and P. acnes treatment increased hepatic TLR2 and TLR4 protein expression (red) from a very low baseline. (C) Hepatic CD68, CD14, TLR2, and TLR4 mRNA responses to each treatment. (D) Representative overlay showing co-localization of TLR2 (red) and F4/80 (green) staining, suggesting predominant expression of TLR2 on hepatic macrophages. Similar results were observed for TLR4. (E) Circulating serum amyloid A (SAA) protein was significantly elevated by orally delivered LPS at 24 h only in HFD-primed mice—baseline SAA (a measure of the acute phase response) was excessively raised in HCD and P. acnes primed mice. (F,G) Hepatic APR markers and apolipoprotein (Apo)-AI were not significantly increased by orally delivered LPS in HCD and P. acnes primed mice, respectively. (H) Mice fed HFD gained significantly more body weight than mice fed normal chow. (I) 4 weeks HFD did not significantly increase CD68 mRNA in abdominal adipose tissue. Orally delivered LPS did not induce inflammatory markers in abdominal adipose tissue (J) , or aorta (K) of HFD-primed mice within 24 h. Error bars shown are SEM. P -values vs. control condition, ANOVA with Dunnett's test.

Journal: Frontiers in Immunology

Article Title: Dietary Toll-Like Receptor Stimulants Promote Hepatic Inflammation and Impair Reverse Cholesterol Transport in Mice via Macrophage-Dependent Interleukin-1 Production

doi: 10.3389/fimmu.2019.01404

Figure Lengend Snippet: Development of an acute murine model of oral-LPS induced APR and dyslipidaemia. Three methods previously reported to increase the sensitivity of mice to circulating LPS were tested, (i) intraperitoneal administration of heat-killed Propionibacterium acnes ( n = 6 per group), (ii) 4 weeks priming with high fat diet (HFD, n = 10 per group) or (iii) 4 weeks priming with high cholesterol diet (HCD), ( n = 12 per group) ( , , ) (A) Hepatic F4/80 +ve (green) cell number was significantly elevated by all three treatments relative to control mice fed normal chow. (B) HCD and P. acnes treatment increased hepatic TLR2 and TLR4 protein expression (red) from a very low baseline. (C) Hepatic CD68, CD14, TLR2, and TLR4 mRNA responses to each treatment. (D) Representative overlay showing co-localization of TLR2 (red) and F4/80 (green) staining, suggesting predominant expression of TLR2 on hepatic macrophages. Similar results were observed for TLR4. (E) Circulating serum amyloid A (SAA) protein was significantly elevated by orally delivered LPS at 24 h only in HFD-primed mice—baseline SAA (a measure of the acute phase response) was excessively raised in HCD and P. acnes primed mice. (F,G) Hepatic APR markers and apolipoprotein (Apo)-AI were not significantly increased by orally delivered LPS in HCD and P. acnes primed mice, respectively. (H) Mice fed HFD gained significantly more body weight than mice fed normal chow. (I) 4 weeks HFD did not significantly increase CD68 mRNA in abdominal adipose tissue. Orally delivered LPS did not induce inflammatory markers in abdominal adipose tissue (J) , or aorta (K) of HFD-primed mice within 24 h. Error bars shown are SEM. P -values vs. control condition, ANOVA with Dunnett's test.

Techniques: Control, Expressing, Staining

Hepatic macrophages are key mediators of the lipid and acute phase responses to ingested LPS. WT mice ( n = 8–11/gp) were fed low-fat diet (LFD) or high-fat diet (HFD) for 4 weeks, with or without treatment with clodronate liposomes to deplete tissue macrophages. (A) F4/80 (green), TLR2 and TLR4 (red) immunofluorescence in livers of mice fed LFD or HFD for 4 weeks. (B) Hepatic acute phase response mRNA markers in saline (Ctrl) or LPS gavaged HFD-primed mice pretreated (or not) with clodronate liposomes (CL), n = 8–11/gp. (C–G) Serum amyloid A (SAA) protein, hepatic ApoAI mRNA and serum reverse cholesterol transport (RCT) markers in the same groups. P -values vs. control condition, ANOVA with Dunnett's test.

Journal: Frontiers in Immunology

Article Title: Dietary Toll-Like Receptor Stimulants Promote Hepatic Inflammation and Impair Reverse Cholesterol Transport in Mice via Macrophage-Dependent Interleukin-1 Production

doi: 10.3389/fimmu.2019.01404

Figure Lengend Snippet: Hepatic macrophages are key mediators of the lipid and acute phase responses to ingested LPS. WT mice ( n = 8–11/gp) were fed low-fat diet (LFD) or high-fat diet (HFD) for 4 weeks, with or without treatment with clodronate liposomes to deplete tissue macrophages. (A) F4/80 (green), TLR2 and TLR4 (red) immunofluorescence in livers of mice fed LFD or HFD for 4 weeks. (B) Hepatic acute phase response mRNA markers in saline (Ctrl) or LPS gavaged HFD-primed mice pretreated (or not) with clodronate liposomes (CL), n = 8–11/gp. (C–G) Serum amyloid A (SAA) protein, hepatic ApoAI mRNA and serum reverse cholesterol transport (RCT) markers in the same groups. P -values vs. control condition, ANOVA with Dunnett's test.

Techniques: Liposomes, Immunofluorescence, Saline, Control

Isolated hepatic macrophages are sensitive to TLR2 and TLR4-stimulants. (A) Immunofluorescence (F4/80, green, DAPI, blue), showed near complete absence of F4/80 + macrophages in liver of mice 3 days after intraperitoneal administration of 1 mg clodronate liposomes ( n = 8–11/gp). (B) Hepatic CD68 mRNA was also significantly reduced by clodronate liposome (CL) treatment. (C) TNF-α production from liver slices of untreated (low-fat diet, LFD), 4 week high-fat diet-primed (HFD), or 4 week HFD primed then CL treated (HFD+CL) mice ( n = 5/gp) cultured with LPS for 24 h. (D) Serum cholesterol levels in mice primed for 4 weeks with HFD, then treated with CL, then orally gavaged with 200 μl saline alone (Ctrl) or 1 mg LPS, revealed no impact of orally delivered LPS on HDL-C in these mice ( n = 11/gp). (E) Pro-inflammatory cytokine production by hepatic macrophages isolated from LFD- or HFD-primed mice treated with medium alone (Ctrl), Pam 3 CSK 4 or LPS ( n = 3–4/gp). (F) Flow cytometry for TLR2 and TLR4 expression by RAW macrophages (positive control cell-line), in comparison with hepatic macrophages isolated from LFD- or HFD-primed mice (representative of 3 exps). Error bars shown are SEM. Pairwise comparisons by Student's T -test.

Journal: Frontiers in Immunology

Article Title: Dietary Toll-Like Receptor Stimulants Promote Hepatic Inflammation and Impair Reverse Cholesterol Transport in Mice via Macrophage-Dependent Interleukin-1 Production

doi: 10.3389/fimmu.2019.01404

Figure Lengend Snippet: Isolated hepatic macrophages are sensitive to TLR2 and TLR4-stimulants. (A) Immunofluorescence (F4/80, green, DAPI, blue), showed near complete absence of F4/80 + macrophages in liver of mice 3 days after intraperitoneal administration of 1 mg clodronate liposomes ( n = 8–11/gp). (B) Hepatic CD68 mRNA was also significantly reduced by clodronate liposome (CL) treatment. (C) TNF-α production from liver slices of untreated (low-fat diet, LFD), 4 week high-fat diet-primed (HFD), or 4 week HFD primed then CL treated (HFD+CL) mice ( n = 5/gp) cultured with LPS for 24 h. (D) Serum cholesterol levels in mice primed for 4 weeks with HFD, then treated with CL, then orally gavaged with 200 μl saline alone (Ctrl) or 1 mg LPS, revealed no impact of orally delivered LPS on HDL-C in these mice ( n = 11/gp). (E) Pro-inflammatory cytokine production by hepatic macrophages isolated from LFD- or HFD-primed mice treated with medium alone (Ctrl), Pam 3 CSK 4 or LPS ( n = 3–4/gp). (F) Flow cytometry for TLR2 and TLR4 expression by RAW macrophages (positive control cell-line), in comparison with hepatic macrophages isolated from LFD- or HFD-primed mice (representative of 3 exps). Error bars shown are SEM. Pairwise comparisons by Student's T -test.

Techniques: Isolation, Immunofluorescence, Liposomes, Cell Culture, Saline, Flow Cytometry, Expressing, Positive Control, Comparison

Interleukin-1β plays a key role in the downregulation of RCT mediators by hepatocytes in response to products secreted by activated hepatic macrophages. (A,B) Human HepG2 hepatocytes increased serum amyloid A (SAA)-1, and reduced ApoAI, mRNA expression significantly in response to IL-1β, but not stimulants of NOD1 (iEDAP), TLR2 (Pam 3 CSK 4 ), or TLR4 ( E. coli LPS). (C) Conditioned medium of HepG2 cells significantly increased the efflux of 3 H-radiolabelled cholesterol from J774 macrophages. (D) The capacity of conditioned media of HepG2 hepatocytes to accept 3 H-radiolabelled cholesterol effluxed from J774 macrophages was reduced significantly by IL-1β, but not by IL-6, TNF-α, or stimulants of NOD1, TLR2, or TLR4. (E) Flow cytometry showed abundant surface TLR2 and TLR4 on human primary monocytes, but no TLR2 and little TLR4 on HepG2 hepatocytes. Murine primary hepatocytes isolated by hepatic perfusion also showed no expression of TLR2 or TLR4, in low-fat diet (LFD) or high-fat diet (HFD)-primed mice (representative of 4 experiments). (F) Kinetics of ApoAI protein expression in HepG2 cells treated with IL-1β determined by Western blot. (G) HepG2 ApoAI mRNA response to conditioned medium of LPS-treated human monocytes with isotype-control or IL-1β neutralizing antibody ( n = 5 exps). (H,I) ApoAI and SAA1 mRNA responses of primary hepatocytes (isolated from n = 6 LFD-fed mice) to inflammatory cytokines (20 ng/ml). (J) ApoAI mRNA responses of primary hepatocytes ( n = 3 HFD-primed mice) to indicated PAMPs (100 ng/ml). Error bars shown are SEM. P -values vs. control condition, Student's T -tests or ANOVA with Dunnett's test.

Journal: Frontiers in Immunology

Article Title: Dietary Toll-Like Receptor Stimulants Promote Hepatic Inflammation and Impair Reverse Cholesterol Transport in Mice via Macrophage-Dependent Interleukin-1 Production

doi: 10.3389/fimmu.2019.01404

Figure Lengend Snippet: Interleukin-1β plays a key role in the downregulation of RCT mediators by hepatocytes in response to products secreted by activated hepatic macrophages. (A,B) Human HepG2 hepatocytes increased serum amyloid A (SAA)-1, and reduced ApoAI, mRNA expression significantly in response to IL-1β, but not stimulants of NOD1 (iEDAP), TLR2 (Pam 3 CSK 4 ), or TLR4 ( E. coli LPS). (C) Conditioned medium of HepG2 cells significantly increased the efflux of 3 H-radiolabelled cholesterol from J774 macrophages. (D) The capacity of conditioned media of HepG2 hepatocytes to accept 3 H-radiolabelled cholesterol effluxed from J774 macrophages was reduced significantly by IL-1β, but not by IL-6, TNF-α, or stimulants of NOD1, TLR2, or TLR4. (E) Flow cytometry showed abundant surface TLR2 and TLR4 on human primary monocytes, but no TLR2 and little TLR4 on HepG2 hepatocytes. Murine primary hepatocytes isolated by hepatic perfusion also showed no expression of TLR2 or TLR4, in low-fat diet (LFD) or high-fat diet (HFD)-primed mice (representative of 4 experiments). (F) Kinetics of ApoAI protein expression in HepG2 cells treated with IL-1β determined by Western blot. (G) HepG2 ApoAI mRNA response to conditioned medium of LPS-treated human monocytes with isotype-control or IL-1β neutralizing antibody ( n = 5 exps). (H,I) ApoAI and SAA1 mRNA responses of primary hepatocytes (isolated from n = 6 LFD-fed mice) to inflammatory cytokines (20 ng/ml). (J) ApoAI mRNA responses of primary hepatocytes ( n = 3 HFD-primed mice) to indicated PAMPs (100 ng/ml). Error bars shown are SEM. P -values vs. control condition, Student's T -tests or ANOVA with Dunnett's test.

Techniques: Expressing, Flow Cytometry, Isolation, Western Blot, Control

Suggested model for the accumulation of PAMPs in foods and their impact on APR and RCT mediators. The enrichment of the food microbiota in proteobacterial species and their subsequent overgrowth can occur in meat and certain other food products when finely chopped and stored at or above refrigeration temperature. This leads to the accumulation of pro-inflammatory bacterial lipopeptides and lipopolysaccharides, which retain their TLR2 and TLR4-stimulating properties even after cooking has killed remaining viable bacteria . These TLR-stimulants may then be absorbed, particularly when intestinal barrier function is impaired, to reach the portal circulation and the liver. Although hepatocytes are insensitive to these stimulants, they may be detected by hepatic macrophages, which secrete IL-1β and other inflammatory cytokines to trigger the APR programme in hepatocytes. This results in reduced serum levels of ApoAI, which is a key mediator of RCT and atherogenesis in mice . Modulation of the food microbiota therefore represents a novel potential target for the modulation of systemic inflammatory markers and cholesterol metabolism.

Journal: Frontiers in Immunology

Article Title: Dietary Toll-Like Receptor Stimulants Promote Hepatic Inflammation and Impair Reverse Cholesterol Transport in Mice via Macrophage-Dependent Interleukin-1 Production

doi: 10.3389/fimmu.2019.01404

Figure Lengend Snippet: Suggested model for the accumulation of PAMPs in foods and their impact on APR and RCT mediators. The enrichment of the food microbiota in proteobacterial species and their subsequent overgrowth can occur in meat and certain other food products when finely chopped and stored at or above refrigeration temperature. This leads to the accumulation of pro-inflammatory bacterial lipopeptides and lipopolysaccharides, which retain their TLR2 and TLR4-stimulating properties even after cooking has killed remaining viable bacteria . These TLR-stimulants may then be absorbed, particularly when intestinal barrier function is impaired, to reach the portal circulation and the liver. Although hepatocytes are insensitive to these stimulants, they may be detected by hepatic macrophages, which secrete IL-1β and other inflammatory cytokines to trigger the APR programme in hepatocytes. This results in reduced serum levels of ApoAI, which is a key mediator of RCT and atherogenesis in mice . Modulation of the food microbiota therefore represents a novel potential target for the modulation of systemic inflammatory markers and cholesterol metabolism.

Techniques: Bacteria

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