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anti mouse tlr4 md 2 antibody  (Thermo Fisher)


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    Structured Review

    Thermo Fisher anti mouse tlr4 md 2 antibody
    The primer has been used for Realtime-PCR.
    Anti Mouse Tlr4 Md 2 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti mouse tlr4 md 2 antibody/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti mouse tlr4 md 2 antibody - by Bioz Stars, 2024-12
    86/100 stars

    Images

    1) Product Images from "Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein"

    Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095935

    The primer has been used for Realtime-PCR.
    Figure Legend Snippet: The primer has been used for Realtime-PCR.

    Techniques Used:

    Human femoral arteries from patients with angiographic atherosclerotic plaques and interal thoracic artery without plaque were assessed by histological and immunochemical analysis. (A). Sections were stained with hematoxylin and eosin. Or immunofluorescence stains of αSMA and TLR4. (B) Immunohistochemical stains of IL-1β, TNFα, MCP-1 and MMP2; counterstained with hematoxylin. H&E is shown at 40×magnification, and immunofluorescence is shown at 1200× magnification B is shown at 200× magnification. Results are representative of 3 independent experiments. The expression levels of IL-1β, TNF-α, MCP-1 and MMP-2 detected by IHC were determined by assessing its staining using software image pro-plus 6.0. The results were showed as integrated optical density (IOD)/area. Three different sections and five different fields in each section have been detected. (n = 3, Mean±SD, **P<0.01 compared with control).
    Figure Legend Snippet: Human femoral arteries from patients with angiographic atherosclerotic plaques and interal thoracic artery without plaque were assessed by histological and immunochemical analysis. (A). Sections were stained with hematoxylin and eosin. Or immunofluorescence stains of αSMA and TLR4. (B) Immunohistochemical stains of IL-1β, TNFα, MCP-1 and MMP2; counterstained with hematoxylin. H&E is shown at 40×magnification, and immunofluorescence is shown at 1200× magnification B is shown at 200× magnification. Results are representative of 3 independent experiments. The expression levels of IL-1β, TNF-α, MCP-1 and MMP-2 detected by IHC were determined by assessing its staining using software image pro-plus 6.0. The results were showed as integrated optical density (IOD)/area. Three different sections and five different fields in each section have been detected. (n = 3, Mean±SD, **P<0.01 compared with control).

    Techniques Used: Staining, Immunofluorescence, Immunohistochemical staining, Expressing, Software

    (A)–(B) Primary SMCs were incubated for increasing amount of time (24, 48 and 96 hrs with 50 ug/mL) and increasing doses (12.5, 25 and 50 ug/mL for 48 hrs) of oxLDL. The expression of TLR4 was detected by Realtime-PCR (A) and Western blotting (B) and quantified by densitometry in 3 independent experiments (A and B) as relative units (TLR4/β-actin). (Mean ± SD, n = 3; *P<0.05, **P<0.01 compared with con group). (C) oxLDL stimulated primary SMCs with increasing doses (12.5, 25 and 50 ug/mL for 24 and 48 hrs). Secretion of IL-1β, TNF-α, MCP-1 and MMP-2 was measured by ELISA and quantified in 3 independent experiments. (Mean ± SD, n = 3;**P<0.01 compared with con group, $$P<0.01 compared with 24 hrs treatment group, ##P<0.01 compared with 12.5 ug/mL oxLDL treatment group, && P<0.01 compared with 25 ug/mL oxLDL).
    Figure Legend Snippet: (A)–(B) Primary SMCs were incubated for increasing amount of time (24, 48 and 96 hrs with 50 ug/mL) and increasing doses (12.5, 25 and 50 ug/mL for 48 hrs) of oxLDL. The expression of TLR4 was detected by Realtime-PCR (A) and Western blotting (B) and quantified by densitometry in 3 independent experiments (A and B) as relative units (TLR4/β-actin). (Mean ± SD, n = 3; *P<0.05, **P<0.01 compared with con group). (C) oxLDL stimulated primary SMCs with increasing doses (12.5, 25 and 50 ug/mL for 24 and 48 hrs). Secretion of IL-1β, TNF-α, MCP-1 and MMP-2 was measured by ELISA and quantified in 3 independent experiments. (Mean ± SD, n = 3;**P<0.01 compared with con group, $$P<0.01 compared with 24 hrs treatment group, ##P<0.01 compared with 12.5 ug/mL oxLDL treatment group, && P<0.01 compared with 25 ug/mL oxLDL).

    Techniques Used: Incubation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

    The primary smooth muscle cells have been cultured from TLR4−/− and C57BL/6 mice. (A) The phenotype of TLR4−/− and C57BL/6 had been detected with Western-blot to assay TLR4 expression. Each type mice choose four different mice. (B) The cells have been incubated with or without oxLDL (50 ug/mL) for 48 hrs, and detected the level of IL-1β, TNF-α, MCP-1 and MMP-2 in the supernatant of cell culture medium. (Mean ± SD, n = 3;**P<0.01 compared with con group, $$P<0.01 compared with C57BL/6 mice group).
    Figure Legend Snippet: The primary smooth muscle cells have been cultured from TLR4−/− and C57BL/6 mice. (A) The phenotype of TLR4−/− and C57BL/6 had been detected with Western-blot to assay TLR4 expression. Each type mice choose four different mice. (B) The cells have been incubated with or without oxLDL (50 ug/mL) for 48 hrs, and detected the level of IL-1β, TNF-α, MCP-1 and MMP-2 in the supernatant of cell culture medium. (Mean ± SD, n = 3;**P<0.01 compared with con group, $$P<0.01 compared with C57BL/6 mice group).

    Techniques Used: Cell Culture, Western Blot, Expressing, Incubation

    The primary smooth muscle cells have been pretreated with TLR4 blocking antibody, with mouse IgG used as control. The cells have been incubated with or without oxLDL (50 ug/mL) for 48 hrs, and levels of IL-1β, TNF-α, MCP-1 and MMP-2 in the supernatant of cell culture medium were measured. (Mean ± SD, n = 3; **P<0.01 compared with con group, $$P<0.01 compared with mouse IgG treatment group).
    Figure Legend Snippet: The primary smooth muscle cells have been pretreated with TLR4 blocking antibody, with mouse IgG used as control. The cells have been incubated with or without oxLDL (50 ug/mL) for 48 hrs, and levels of IL-1β, TNF-α, MCP-1 and MMP-2 in the supernatant of cell culture medium were measured. (Mean ± SD, n = 3; **P<0.01 compared with con group, $$P<0.01 compared with mouse IgG treatment group).

    Techniques Used: Blocking Assay, Incubation, Cell Culture

    (A)–(C) SMCs were incubated for increasing amount of time (30, 60,120 and 240 min with 50 ug/mL)of oxLDL. The phosphorylation of p38 and NF-κB was detected by Western blotting (A) and quantified by densitometry in 3 independent experiments (B and C) as relative units (p38 or NF-κB phosphorylated protein/total protein). (Mean ± SD, n = 3; *P<0.05, **P<0.01 compared with con group). (D)- (G) The primary SMCs of TLR4 −/− (C57BL/6 as control) and TLR4 antibody blocking (mouse IgG as control) have been treated with or without oxLDL (50 ug/mL) for 240 min to measure the activation of p38 or NF-κB in 3 independent experiments as relative units (p38 or NF-κB phosphorylated protein/total protein). (Mean ± SD, n = 3; **P<0.01 compared with oxLDL untreated group, ##P<0.01 compared with C57BL/6 group, $P<0.05, $$P<0.01 compared with mouse IgG group).
    Figure Legend Snippet: (A)–(C) SMCs were incubated for increasing amount of time (30, 60,120 and 240 min with 50 ug/mL)of oxLDL. The phosphorylation of p38 and NF-κB was detected by Western blotting (A) and quantified by densitometry in 3 independent experiments (B and C) as relative units (p38 or NF-κB phosphorylated protein/total protein). (Mean ± SD, n = 3; *P<0.05, **P<0.01 compared with con group). (D)- (G) The primary SMCs of TLR4 −/− (C57BL/6 as control) and TLR4 antibody blocking (mouse IgG as control) have been treated with or without oxLDL (50 ug/mL) for 240 min to measure the activation of p38 or NF-κB in 3 independent experiments as relative units (p38 or NF-κB phosphorylated protein/total protein). (Mean ± SD, n = 3; **P<0.01 compared with oxLDL untreated group, ##P<0.01 compared with C57BL/6 group, $P<0.05, $$P<0.01 compared with mouse IgG group).

    Techniques Used: Incubation, Western Blot, Blocking Assay, Activation Assay



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    Image Search Results


    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Candida albicans oscillating UME6 expression during intestinal colonization primes systemic Th17 protective immunity

    doi: 10.1016/j.celrep.2022.110837

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Anti-mouse TLR4/MD2 (clone MTS510) , Hycult Biotech , Cat#HM1029; RRID:AB_533218.

    Techniques: Recombinant, Staining, Conjugation Assay, SYBR Green Assay, Software

    The primer has been used for Realtime-PCR.

    Journal: PLoS ONE

    Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

    doi: 10.1371/journal.pone.0095935

    Figure Lengend Snippet: The primer has been used for Realtime-PCR.

    Article Snippet: Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control.

    Techniques:

    Human femoral arteries from patients with angiographic atherosclerotic plaques and interal thoracic artery without plaque were assessed by histological and immunochemical analysis. (A). Sections were stained with hematoxylin and eosin. Or immunofluorescence stains of αSMA and TLR4. (B) Immunohistochemical stains of IL-1β, TNFα, MCP-1 and MMP2; counterstained with hematoxylin. H&E is shown at 40×magnification, and immunofluorescence is shown at 1200× magnification B is shown at 200× magnification. Results are representative of 3 independent experiments. The expression levels of IL-1β, TNF-α, MCP-1 and MMP-2 detected by IHC were determined by assessing its staining using software image pro-plus 6.0. The results were showed as integrated optical density (IOD)/area. Three different sections and five different fields in each section have been detected. (n = 3, Mean±SD, **P<0.01 compared with control).

    Journal: PLoS ONE

    Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

    doi: 10.1371/journal.pone.0095935

    Figure Lengend Snippet: Human femoral arteries from patients with angiographic atherosclerotic plaques and interal thoracic artery without plaque were assessed by histological and immunochemical analysis. (A). Sections were stained with hematoxylin and eosin. Or immunofluorescence stains of αSMA and TLR4. (B) Immunohistochemical stains of IL-1β, TNFα, MCP-1 and MMP2; counterstained with hematoxylin. H&E is shown at 40×magnification, and immunofluorescence is shown at 1200× magnification B is shown at 200× magnification. Results are representative of 3 independent experiments. The expression levels of IL-1β, TNF-α, MCP-1 and MMP-2 detected by IHC were determined by assessing its staining using software image pro-plus 6.0. The results were showed as integrated optical density (IOD)/area. Three different sections and five different fields in each section have been detected. (n = 3, Mean±SD, **P<0.01 compared with control).

    Article Snippet: Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control.

    Techniques: Staining, Immunofluorescence, Immunohistochemical staining, Expressing, Software

    (A)–(B) Primary SMCs were incubated for increasing amount of time (24, 48 and 96 hrs with 50 ug/mL) and increasing doses (12.5, 25 and 50 ug/mL for 48 hrs) of oxLDL. The expression of TLR4 was detected by Realtime-PCR (A) and Western blotting (B) and quantified by densitometry in 3 independent experiments (A and B) as relative units (TLR4/β-actin). (Mean ± SD, n = 3; *P<0.05, **P<0.01 compared with con group). (C) oxLDL stimulated primary SMCs with increasing doses (12.5, 25 and 50 ug/mL for 24 and 48 hrs). Secretion of IL-1β, TNF-α, MCP-1 and MMP-2 was measured by ELISA and quantified in 3 independent experiments. (Mean ± SD, n = 3;**P<0.01 compared with con group, $$P<0.01 compared with 24 hrs treatment group, ##P<0.01 compared with 12.5 ug/mL oxLDL treatment group, && P<0.01 compared with 25 ug/mL oxLDL).

    Journal: PLoS ONE

    Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

    doi: 10.1371/journal.pone.0095935

    Figure Lengend Snippet: (A)–(B) Primary SMCs were incubated for increasing amount of time (24, 48 and 96 hrs with 50 ug/mL) and increasing doses (12.5, 25 and 50 ug/mL for 48 hrs) of oxLDL. The expression of TLR4 was detected by Realtime-PCR (A) and Western blotting (B) and quantified by densitometry in 3 independent experiments (A and B) as relative units (TLR4/β-actin). (Mean ± SD, n = 3; *P<0.05, **P<0.01 compared with con group). (C) oxLDL stimulated primary SMCs with increasing doses (12.5, 25 and 50 ug/mL for 24 and 48 hrs). Secretion of IL-1β, TNF-α, MCP-1 and MMP-2 was measured by ELISA and quantified in 3 independent experiments. (Mean ± SD, n = 3;**P<0.01 compared with con group, $$P<0.01 compared with 24 hrs treatment group, ##P<0.01 compared with 12.5 ug/mL oxLDL treatment group, && P<0.01 compared with 25 ug/mL oxLDL).

    Article Snippet: Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control.

    Techniques: Incubation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

    The primary smooth muscle cells have been cultured from TLR4−/− and C57BL/6 mice. (A) The phenotype of TLR4−/− and C57BL/6 had been detected with Western-blot to assay TLR4 expression. Each type mice choose four different mice. (B) The cells have been incubated with or without oxLDL (50 ug/mL) for 48 hrs, and detected the level of IL-1β, TNF-α, MCP-1 and MMP-2 in the supernatant of cell culture medium. (Mean ± SD, n = 3;**P<0.01 compared with con group, $$P<0.01 compared with C57BL/6 mice group).

    Journal: PLoS ONE

    Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

    doi: 10.1371/journal.pone.0095935

    Figure Lengend Snippet: The primary smooth muscle cells have been cultured from TLR4−/− and C57BL/6 mice. (A) The phenotype of TLR4−/− and C57BL/6 had been detected with Western-blot to assay TLR4 expression. Each type mice choose four different mice. (B) The cells have been incubated with or without oxLDL (50 ug/mL) for 48 hrs, and detected the level of IL-1β, TNF-α, MCP-1 and MMP-2 in the supernatant of cell culture medium. (Mean ± SD, n = 3;**P<0.01 compared with con group, $$P<0.01 compared with C57BL/6 mice group).

    Article Snippet: Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control.

    Techniques: Cell Culture, Western Blot, Expressing, Incubation

    The primary smooth muscle cells have been pretreated with TLR4 blocking antibody, with mouse IgG used as control. The cells have been incubated with or without oxLDL (50 ug/mL) for 48 hrs, and levels of IL-1β, TNF-α, MCP-1 and MMP-2 in the supernatant of cell culture medium were measured. (Mean ± SD, n = 3; **P<0.01 compared with con group, $$P<0.01 compared with mouse IgG treatment group).

    Journal: PLoS ONE

    Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

    doi: 10.1371/journal.pone.0095935

    Figure Lengend Snippet: The primary smooth muscle cells have been pretreated with TLR4 blocking antibody, with mouse IgG used as control. The cells have been incubated with or without oxLDL (50 ug/mL) for 48 hrs, and levels of IL-1β, TNF-α, MCP-1 and MMP-2 in the supernatant of cell culture medium were measured. (Mean ± SD, n = 3; **P<0.01 compared with con group, $$P<0.01 compared with mouse IgG treatment group).

    Article Snippet: Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control.

    Techniques: Blocking Assay, Incubation, Cell Culture

    (A)–(C) SMCs were incubated for increasing amount of time (30, 60,120 and 240 min with 50 ug/mL)of oxLDL. The phosphorylation of p38 and NF-κB was detected by Western blotting (A) and quantified by densitometry in 3 independent experiments (B and C) as relative units (p38 or NF-κB phosphorylated protein/total protein). (Mean ± SD, n = 3; *P<0.05, **P<0.01 compared with con group). (D)- (G) The primary SMCs of TLR4 −/− (C57BL/6 as control) and TLR4 antibody blocking (mouse IgG as control) have been treated with or without oxLDL (50 ug/mL) for 240 min to measure the activation of p38 or NF-κB in 3 independent experiments as relative units (p38 or NF-κB phosphorylated protein/total protein). (Mean ± SD, n = 3; **P<0.01 compared with oxLDL untreated group, ##P<0.01 compared with C57BL/6 group, $P<0.05, $$P<0.01 compared with mouse IgG group).

    Journal: PLoS ONE

    Article Title: Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

    doi: 10.1371/journal.pone.0095935

    Figure Lengend Snippet: (A)–(C) SMCs were incubated for increasing amount of time (30, 60,120 and 240 min with 50 ug/mL)of oxLDL. The phosphorylation of p38 and NF-κB was detected by Western blotting (A) and quantified by densitometry in 3 independent experiments (B and C) as relative units (p38 or NF-κB phosphorylated protein/total protein). (Mean ± SD, n = 3; *P<0.05, **P<0.01 compared with con group). (D)- (G) The primary SMCs of TLR4 −/− (C57BL/6 as control) and TLR4 antibody blocking (mouse IgG as control) have been treated with or without oxLDL (50 ug/mL) for 240 min to measure the activation of p38 or NF-κB in 3 independent experiments as relative units (p38 or NF-κB phosphorylated protein/total protein). (Mean ± SD, n = 3; **P<0.01 compared with oxLDL untreated group, ##P<0.01 compared with C57BL/6 group, $P<0.05, $$P<0.01 compared with mouse IgG group).

    Article Snippet: Activation of TLR4 was blocked by Anti-mouse TLR4/MD-2 antibody (eBioscience, 16–9924, USA) and normal mouse IgG (eBioscience, USA) was used as negative control.

    Techniques: Incubation, Western Blot, Blocking Assay, Activation Assay

    Primary antibodies

    Journal: The Journal of Experimental Medicine

    Article Title: Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF

    doi: 10.1084/jem.20210862

    Figure Lengend Snippet: Primary antibodies

    Article Snippet: TLR4 , BioRad , MCA2154T.

    Techniques:

    TLR4 + immune cells induce epithelial NF-κB signaling in the cecal mucosa upon LPS exposure. Mice were i.v. injected with LPS. Cecal explants were imaged at 1 h.p.inj. by two-photon microscopy. (A–C) Representative images and quantification of epithelial NF-κB activation in the indicated mice (A, n = 4–7) or BMCs (B, n = 5; C, n = 5). Each circle represents one mouse. Black line: median. **, P ≤ 0.01 by Mann–Whitney U test. (D) Small intestinal organoids were treated with 5, 50, or 500 ng/ml LPS and imaged for ∼1 h. Representative images of one organoid over time (top) and quantification of NF-κB activation (bottom; relative change). Each circle represents one organoid at the given time (minutes after start of the treatment, n = 7). Lines connect data points from the same organoid. Red dashed line: 50% activation threshold. Black dotted line: no change. Scale bars: 50 µm. Combined data of two (C and D), three (A), or four (B) independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF

    doi: 10.1084/jem.20210862

    Figure Lengend Snippet: TLR4 + immune cells induce epithelial NF-κB signaling in the cecal mucosa upon LPS exposure. Mice were i.v. injected with LPS. Cecal explants were imaged at 1 h.p.inj. by two-photon microscopy. (A–C) Representative images and quantification of epithelial NF-κB activation in the indicated mice (A, n = 4–7) or BMCs (B, n = 5; C, n = 5). Each circle represents one mouse. Black line: median. **, P ≤ 0.01 by Mann–Whitney U test. (D) Small intestinal organoids were treated with 5, 50, or 500 ng/ml LPS and imaged for ∼1 h. Representative images of one organoid over time (top) and quantification of NF-κB activation (bottom; relative change). Each circle represents one organoid at the given time (minutes after start of the treatment, n = 7). Lines connect data points from the same organoid. Red dashed line: 50% activation threshold. Black dotted line: no change. Scale bars: 50 µm. Combined data of two (C and D), three (A), or four (B) independent experiments.

    Article Snippet: TLR4 , BioRad , MCA2154T.

    Techniques: Injection, Microscopy, Activation Assay, MANN-WHITNEY

    TLR4 + immune cells induce epithelial NF-κB activation in the small intestine and colon. (A) Schematic drawing of the two-photon imaging (left). The intestinal mucosa is imaged from the luminal side (black arrowhead), resulting in images in horizontal plane of the mucosa (right; part of the image shown in ). White dashed line/E, epithelium; L, lumen; white asterisks, epithelial nuclei. (B) Fold changes in expression of A20 , Cxcl2 , and Tnf in the cecal mucosa of mice depicted in ( n = 5). (C) Fold changes in expression of A20 , Cxcl2 , and Tnf in the cecal mucosa of mice depicted in in comparison to PBS-injected mice in ( n = 5). (D and E) Two-photon microscopy images and quantification of epithelial NF-κB activation in the (D; n = 3–5) small intestine and (E; n = 3–5) colon of LPS-injected BMCs, and (F) in small intestine and colon of Myd88 −/− > p65 GFP-FL x Tlr4 −/− , Ticam1 −/− > p65 GFP-FL x Tlr4 −/− BMCs. Each circle represents one mouse. Black line: median. Statistical analysis: Mann–Whitney U test. *, P ≤ 0.05; **, ≤ 0.01. Scale bars: 50 µm. Combined data of three (B), four (C), five (D), six (E), or seven (F) independent experiments. Each circle represents one mouse. Black line: median. Scale bars: 50 µm.

    Journal: The Journal of Experimental Medicine

    Article Title: Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF

    doi: 10.1084/jem.20210862

    Figure Lengend Snippet: TLR4 + immune cells induce epithelial NF-κB activation in the small intestine and colon. (A) Schematic drawing of the two-photon imaging (left). The intestinal mucosa is imaged from the luminal side (black arrowhead), resulting in images in horizontal plane of the mucosa (right; part of the image shown in ). White dashed line/E, epithelium; L, lumen; white asterisks, epithelial nuclei. (B) Fold changes in expression of A20 , Cxcl2 , and Tnf in the cecal mucosa of mice depicted in ( n = 5). (C) Fold changes in expression of A20 , Cxcl2 , and Tnf in the cecal mucosa of mice depicted in in comparison to PBS-injected mice in ( n = 5). (D and E) Two-photon microscopy images and quantification of epithelial NF-κB activation in the (D; n = 3–5) small intestine and (E; n = 3–5) colon of LPS-injected BMCs, and (F) in small intestine and colon of Myd88 −/− > p65 GFP-FL x Tlr4 −/− , Ticam1 −/− > p65 GFP-FL x Tlr4 −/− BMCs. Each circle represents one mouse. Black line: median. Statistical analysis: Mann–Whitney U test. *, P ≤ 0.05; **, ≤ 0.01. Scale bars: 50 µm. Combined data of three (B), four (C), five (D), six (E), or seven (F) independent experiments. Each circle represents one mouse. Black line: median. Scale bars: 50 µm.

    Article Snippet: TLR4 , BioRad , MCA2154T.

    Techniques: Activation Assay, Imaging, Expressing, Injection, Microscopy, MANN-WHITNEY

    TNF produced by CD11c + cells induces local epithelial NF-κB activation in the intestinal mucosa. (A and B) p65 GFP-FL intestinal epithelial organoids established from the indicated regions were treated with 5, 50, and 500 ng/ml or 5 µg/ml LPS (+ LBP and CD14, if indicated) and imaged for 1 h (A; n = 3–17), or analyzed by qPCR at 3 h of treatment (B and C; n = 6 or 7). (C) Colon organoids from p65 GFP-FL x Tlr4 −/− mice ( n = 4). (D) Representative two-photon microscopy overview image of the cecal mucosa of mice described in at 1 h.p.inj. of LPS ( n = 6). Red squares indicate RFP + ( Tlr4 +/+ ) cells. White lines indicate IEC NF-κB activation zones (defined as areas with continuous epithelial NF-κB activation). (E) Quantification of epithelial NF-κB activation in Il18 −/− > p65 GFP-FL x Tlr4 −/− , Il18r −/− > p65 GFP-FL x Tlr4 −/− , and Il1ab −/− > p65 GFP-FL x Tlr4 −/− BMCs at 1 h.p.inj. of LPS ( n = 7 or 8). (F) p65 GFP-FL intestinal epithelial organoids from cecum (left) or colon (right) were treated with 5, 50, and 500 ng/ml TNF and imaged for 1 h ( n = 3–17). (G) Quantification of epithelial NF-κB activation in mice as described in . Mice pretreated with DTX were injected with PBS or TNF ( n = 2–6). Cecae were imaged at 1 h.p.inj. Data of LPS-injected mice are replotted from for comparison. Black line: median (B, C, E, and G). Dashed line: detection limit (C and G) or error range (A and F). Each circle represents one organoid sample (B and C), one mouse (E and G), or the median (A and F). Statistical analysis: one-way ANOVA with Dunett’s correction (B and C) or Mann–Whitney U test (E and G). *, P ≤ 0.05; **, P ≤ 0.01. Scale bars: 50 µm. Combined data of two (A, small intestine; B, C, D, and F, cecum), three (A, cecum), four (F, colon), six (B and E), or eight (A, colon) independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF

    doi: 10.1084/jem.20210862

    Figure Lengend Snippet: TNF produced by CD11c + cells induces local epithelial NF-κB activation in the intestinal mucosa. (A and B) p65 GFP-FL intestinal epithelial organoids established from the indicated regions were treated with 5, 50, and 500 ng/ml or 5 µg/ml LPS (+ LBP and CD14, if indicated) and imaged for 1 h (A; n = 3–17), or analyzed by qPCR at 3 h of treatment (B and C; n = 6 or 7). (C) Colon organoids from p65 GFP-FL x Tlr4 −/− mice ( n = 4). (D) Representative two-photon microscopy overview image of the cecal mucosa of mice described in at 1 h.p.inj. of LPS ( n = 6). Red squares indicate RFP + ( Tlr4 +/+ ) cells. White lines indicate IEC NF-κB activation zones (defined as areas with continuous epithelial NF-κB activation). (E) Quantification of epithelial NF-κB activation in Il18 −/− > p65 GFP-FL x Tlr4 −/− , Il18r −/− > p65 GFP-FL x Tlr4 −/− , and Il1ab −/− > p65 GFP-FL x Tlr4 −/− BMCs at 1 h.p.inj. of LPS ( n = 7 or 8). (F) p65 GFP-FL intestinal epithelial organoids from cecum (left) or colon (right) were treated with 5, 50, and 500 ng/ml TNF and imaged for 1 h ( n = 3–17). (G) Quantification of epithelial NF-κB activation in mice as described in . Mice pretreated with DTX were injected with PBS or TNF ( n = 2–6). Cecae were imaged at 1 h.p.inj. Data of LPS-injected mice are replotted from for comparison. Black line: median (B, C, E, and G). Dashed line: detection limit (C and G) or error range (A and F). Each circle represents one organoid sample (B and C), one mouse (E and G), or the median (A and F). Statistical analysis: one-way ANOVA with Dunett’s correction (B and C) or Mann–Whitney U test (E and G). *, P ≤ 0.05; **, P ≤ 0.01. Scale bars: 50 µm. Combined data of two (A, small intestine; B, C, D, and F, cecum), three (A, cecum), four (F, colon), six (B and E), or eight (A, colon) independent experiments.

    Article Snippet: TLR4 , BioRad , MCA2154T.

    Techniques: Produced, Activation Assay, Microscopy, Injection, MANN-WHITNEY

    CD11c + cells induce local epithelial NF-κB activation via TNF. Mice were i.v. injected with LPS and cecal explants imaged at 1 h.p.inj. by two-photon microscopy (representative image and quantification) if not indicated otherwise. (A) Cecum mucosa from p65 GFP-FL x Tlr4 −/− mice reconstituted with a 1:10 mix of ActRFP (10%, Tlr4 +/+ ) and p65 GFP-FL x Tlr4 −/− (90%) BM. Analysis of RFP + cells within an epithelial NF-κB activation zone (see , n = 10–18). (B – D) Cecal epithelium NF-κB activation of the indicated BMCs or p65 GFP-FL mice pretreated with isotype control/anti-TNF antibody or i.v. injected with TNF and analyzed at the indicated time points ( n = 5 or 6). (E) TNF-treated small-intestinal epithelial organoids. Representative image and quantification of NF-κB activation kinetics with 5, 50, or 500 ng/ml TNF ( n = 9–17). Lines connect data points from the same organoid. Red dashed line: 50% activation threshold. Black dotted line: no change. (F) Representative images of the cecal epithelium and quantification of epithelial NF-κB activation of p65 GFP-FL x Tlr4 −/− mice reconstituted with a 1:20 mix of CD11c-DTR and TNFa −/− BM, pretreated with DTX ( n = 5–8). (B–D and F) Black line: median. *, P ≤ 0.05; **, P ≤ 0.01 by Mann–Whitney U test. Each circle represents one mouse or one organoid (E). Combined data of two (A and B), three (D and E), four (F), or six (C) independent experiments. Scale bars: 50 µm.

    Journal: The Journal of Experimental Medicine

    Article Title: Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF

    doi: 10.1084/jem.20210862

    Figure Lengend Snippet: CD11c + cells induce local epithelial NF-κB activation via TNF. Mice were i.v. injected with LPS and cecal explants imaged at 1 h.p.inj. by two-photon microscopy (representative image and quantification) if not indicated otherwise. (A) Cecum mucosa from p65 GFP-FL x Tlr4 −/− mice reconstituted with a 1:10 mix of ActRFP (10%, Tlr4 +/+ ) and p65 GFP-FL x Tlr4 −/− (90%) BM. Analysis of RFP + cells within an epithelial NF-κB activation zone (see , n = 10–18). (B – D) Cecal epithelium NF-κB activation of the indicated BMCs or p65 GFP-FL mice pretreated with isotype control/anti-TNF antibody or i.v. injected with TNF and analyzed at the indicated time points ( n = 5 or 6). (E) TNF-treated small-intestinal epithelial organoids. Representative image and quantification of NF-κB activation kinetics with 5, 50, or 500 ng/ml TNF ( n = 9–17). Lines connect data points from the same organoid. Red dashed line: 50% activation threshold. Black dotted line: no change. (F) Representative images of the cecal epithelium and quantification of epithelial NF-κB activation of p65 GFP-FL x Tlr4 −/− mice reconstituted with a 1:20 mix of CD11c-DTR and TNFa −/− BM, pretreated with DTX ( n = 5–8). (B–D and F) Black line: median. *, P ≤ 0.05; **, P ≤ 0.01 by Mann–Whitney U test. Each circle represents one mouse or one organoid (E). Combined data of two (A and B), three (D and E), four (F), or six (C) independent experiments. Scale bars: 50 µm.

    Article Snippet: TLR4 , BioRad , MCA2154T.

    Techniques: Activation Assay, Injection, Microscopy, MANN-WHITNEY

    Receptor expression in IECs. (A–D) Confocal microscopy images of (A) the cecal patch, and (B) a mucosa-associated lymphoid follicle in fixed cecae of p65 GFP-FL mice i.v. injected with LPS at 1 h.p.inj. Boxes in overview images indicate insets. Arrowheads indicate p65 + nuclei. Arrows indicate p65 − nuclei. Scale bars: 50 µm (overview images) or 10 µm (insets). TLR4 staining in small intestine (C) and colon (D) of WT mice. Arrowheads indicate MHCII + cells. Arrows indicate IECs. Scale bars: 20 µm. Representative images of mice from two experiments. (E) Heat map depicting expression levels of Tlr2, Tlr4, Tlr6, Tlr11 , Tnfrsf1a (TNFR1), and Tnfrsf1b (TNFR2) in untreated or TNF-treated (5 ng/ml, 8 h) small intestinal epithelial organoids derived from SPF (SPF1, SPF2) or germ-free (GF) mice, m-IC c12 cells, and mouse embryonic fibroblasts (MEFs; reanalysis of a previously published transcriptome dataset, all detectable Tlrs depicted; ). (F) Cecal mucosa stained for TNFR1 at 1 h.p.inj. of LPS. Scale bars: 50 µm. Representative images of mice from three independent experiments ( n = 4–7).

    Journal: The Journal of Experimental Medicine

    Article Title: Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF

    doi: 10.1084/jem.20210862

    Figure Lengend Snippet: Receptor expression in IECs. (A–D) Confocal microscopy images of (A) the cecal patch, and (B) a mucosa-associated lymphoid follicle in fixed cecae of p65 GFP-FL mice i.v. injected with LPS at 1 h.p.inj. Boxes in overview images indicate insets. Arrowheads indicate p65 + nuclei. Arrows indicate p65 − nuclei. Scale bars: 50 µm (overview images) or 10 µm (insets). TLR4 staining in small intestine (C) and colon (D) of WT mice. Arrowheads indicate MHCII + cells. Arrows indicate IECs. Scale bars: 20 µm. Representative images of mice from two experiments. (E) Heat map depicting expression levels of Tlr2, Tlr4, Tlr6, Tlr11 , Tnfrsf1a (TNFR1), and Tnfrsf1b (TNFR2) in untreated or TNF-treated (5 ng/ml, 8 h) small intestinal epithelial organoids derived from SPF (SPF1, SPF2) or germ-free (GF) mice, m-IC c12 cells, and mouse embryonic fibroblasts (MEFs; reanalysis of a previously published transcriptome dataset, all detectable Tlrs depicted; ). (F) Cecal mucosa stained for TNFR1 at 1 h.p.inj. of LPS. Scale bars: 50 µm. Representative images of mice from three independent experiments ( n = 4–7).

    Article Snippet: TLR4 , BioRad , MCA2154T.

    Techniques: Expressing, Confocal Microscopy, Injection, Staining, Derivative Assay

    Tissue resident, monocyte-derived macrophages secrete TNF to induce local epithelial NF-κB activation. (A) ELISA measurements of TNF concentrations in the cecal mucosa of LPS injected WT mice ( n = 5 or 6). Dashed line: detection limit. y axis in log 10 scale. (B) Percentage of TNF + DCs or macrophages (gating as shown in ) in the cecum, small intestine, and colon of LPS-treated WT mice (1 h.p.inj.) and PBS-treated controls ( n = 5–7). (C) Representative images of the cecal epithelium and quantification of epithelial NF-κB activation of p65 GFP-FL mice pretreated with anti-CSF1R or isotype control, injected with LPS, and imaged 1 h.p.inj. ( n = 7). Depletion efficiency of macrophages and DCs in anti-CSF1R treated mice. (D) Normalized marker expression of TNF + compared with TNF − macrophages in the cecum, small intestine, and colon of LPS-injected WT mice ( n = 4–7). (E) Percentage of CD4 +/− Tim4 +/− cells among TNF − and TNF + macrophages in the cecum of LPS-injected WT mice ( n = 4). (F) TNF-PLA analysis of cecae from p65 GFP-FL x Tlr4 −/− mice reconstituted with a 1:40 mix of ActRFP (2.5%, Tlr4 +/+ ) and p65 GFP-FL x Tlr4 −/− (97.5%) BM. Representative confocal microscopy image of fixed cecal tissue at 40 min.p.inj. (left) and quantification of PLA for TNF in crypts without (−) or with (+) epithelial NF-κB activation at 1 h.p.inj. ( n = 11–13). Scale bar: 10 µm. Black line: median. Statistical analysis: one-way ANOVA with Dunett’s correction (A), two-way ANOVA with Sidak’s multiple comparison test (B), or Mann–Whitney U test (C, E, and F). *, P ≤ 0.05; **, P ≤ 0.01. Each circle represents one mouse (A–E) or one crypt (F; five mice analyzed). Combined data of two (D), three (B and C), four (F), or six (A) independent experiments, or exemplary data of two (E) independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF

    doi: 10.1084/jem.20210862

    Figure Lengend Snippet: Tissue resident, monocyte-derived macrophages secrete TNF to induce local epithelial NF-κB activation. (A) ELISA measurements of TNF concentrations in the cecal mucosa of LPS injected WT mice ( n = 5 or 6). Dashed line: detection limit. y axis in log 10 scale. (B) Percentage of TNF + DCs or macrophages (gating as shown in ) in the cecum, small intestine, and colon of LPS-treated WT mice (1 h.p.inj.) and PBS-treated controls ( n = 5–7). (C) Representative images of the cecal epithelium and quantification of epithelial NF-κB activation of p65 GFP-FL mice pretreated with anti-CSF1R or isotype control, injected with LPS, and imaged 1 h.p.inj. ( n = 7). Depletion efficiency of macrophages and DCs in anti-CSF1R treated mice. (D) Normalized marker expression of TNF + compared with TNF − macrophages in the cecum, small intestine, and colon of LPS-injected WT mice ( n = 4–7). (E) Percentage of CD4 +/− Tim4 +/− cells among TNF − and TNF + macrophages in the cecum of LPS-injected WT mice ( n = 4). (F) TNF-PLA analysis of cecae from p65 GFP-FL x Tlr4 −/− mice reconstituted with a 1:40 mix of ActRFP (2.5%, Tlr4 +/+ ) and p65 GFP-FL x Tlr4 −/− (97.5%) BM. Representative confocal microscopy image of fixed cecal tissue at 40 min.p.inj. (left) and quantification of PLA for TNF in crypts without (−) or with (+) epithelial NF-κB activation at 1 h.p.inj. ( n = 11–13). Scale bar: 10 µm. Black line: median. Statistical analysis: one-way ANOVA with Dunett’s correction (A), two-way ANOVA with Sidak’s multiple comparison test (B), or Mann–Whitney U test (C, E, and F). *, P ≤ 0.05; **, P ≤ 0.01. Each circle represents one mouse (A–E) or one crypt (F; five mice analyzed). Combined data of two (D), three (B and C), four (F), or six (A) independent experiments, or exemplary data of two (E) independent experiments.

    Article Snippet: TLR4 , BioRad , MCA2154T.

    Techniques: Derivative Assay, Activation Assay, Enzyme-linked Immunosorbent Assay, Injection, Marker, Expressing, Confocal Microscopy, MANN-WHITNEY

    Intestinal macrophages secrete TNF to induce local epithelial NF-κB activation. (A) Gating strategy for intestinal MP subsets in the cecal mucosa of mice depicted in B and C. (B and C) Flow cytometry analysis of cecal MPs from PBS- or LPS-injected (B) KappaBle mice for assessment of NF-κB activation (gating as shown in A; n = 3–5) or (C) WT mice for identification of TNF-producing MP subsets (gating as shown in A; n = 3–6). (D) Updated gating strategy for differentiation of intestinal DCs and macrophages as shown in . Lineage = NK1.1, CD3, B220. (E) For scRNAseq, CD45 + live MHCII + lineage (NK1.1, CD3, B220) − cells were sorted from the cecal mucosa of 40 min LPS-injected mice or PBS-treated controls ( n = 4 mice) and subsequently analyzed by scRNAseq (10X Genomics). T-distributed stochastic neighbor embedding plots showing the distribution of the analyzed cells indicated by cluster (left) or treatment (right). (F) Expression levels of intestinal MP markers: this analysis revealed two clear macrophages clusters (7 and 10). CD11b − CD103 + Xcr1 + DCs were represented in clusters 1–4, out of which cluster 2 mainly consisted of cells from LPS-treated mice, indicating that this might represent an activated state. This is in line with the secondary TNF production of this subset at later time points after injection, as detected by flow cytometry (C). Clusters 0 and 5 were positive for Sirpa and contained CD11b + CD103 + DCs (cluster 0) and CD11b + CD103 − DCs (cluster 5). While clusters 12 and 13 were positive for a number of monocyte/macrophage markers and therefore likely represent maturing macrophages, the assignment of clusters 6, 8, and 9 was challenging due to overlapping marker expression. These clusters, together with cluster 11 (mast cells), likely contained precursors (macrophage/DC, cluster 8) or contaminating cells (T cells, cluster 6 and 8; plasma cells, cluster 9). (G) Expression analysis of TLR4 signaling–associated genes. (H) Gene set enrichment analysis for macrophage clusters (7 and 10). (I) Frequency of DCs and macrophages in the cecum, small intestine, and colon of naive WT mice ( n = 5). Each circle represents one mouse (B, C, and I) or one cell (E). Black line: median. Combined data of two (I), three (B), or nine (C) independent experiments. Statistical analysis: one-way ANOVA with Tukey’s correction (C) or Mann–Whitney U test (B). *, P ≤ 0.05; **, P ≤ 0.01.

    Journal: The Journal of Experimental Medicine

    Article Title: Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF

    doi: 10.1084/jem.20210862

    Figure Lengend Snippet: Intestinal macrophages secrete TNF to induce local epithelial NF-κB activation. (A) Gating strategy for intestinal MP subsets in the cecal mucosa of mice depicted in B and C. (B and C) Flow cytometry analysis of cecal MPs from PBS- or LPS-injected (B) KappaBle mice for assessment of NF-κB activation (gating as shown in A; n = 3–5) or (C) WT mice for identification of TNF-producing MP subsets (gating as shown in A; n = 3–6). (D) Updated gating strategy for differentiation of intestinal DCs and macrophages as shown in . Lineage = NK1.1, CD3, B220. (E) For scRNAseq, CD45 + live MHCII + lineage (NK1.1, CD3, B220) − cells were sorted from the cecal mucosa of 40 min LPS-injected mice or PBS-treated controls ( n = 4 mice) and subsequently analyzed by scRNAseq (10X Genomics). T-distributed stochastic neighbor embedding plots showing the distribution of the analyzed cells indicated by cluster (left) or treatment (right). (F) Expression levels of intestinal MP markers: this analysis revealed two clear macrophages clusters (7 and 10). CD11b − CD103 + Xcr1 + DCs were represented in clusters 1–4, out of which cluster 2 mainly consisted of cells from LPS-treated mice, indicating that this might represent an activated state. This is in line with the secondary TNF production of this subset at later time points after injection, as detected by flow cytometry (C). Clusters 0 and 5 were positive for Sirpa and contained CD11b + CD103 + DCs (cluster 0) and CD11b + CD103 − DCs (cluster 5). While clusters 12 and 13 were positive for a number of monocyte/macrophage markers and therefore likely represent maturing macrophages, the assignment of clusters 6, 8, and 9 was challenging due to overlapping marker expression. These clusters, together with cluster 11 (mast cells), likely contained precursors (macrophage/DC, cluster 8) or contaminating cells (T cells, cluster 6 and 8; plasma cells, cluster 9). (G) Expression analysis of TLR4 signaling–associated genes. (H) Gene set enrichment analysis for macrophage clusters (7 and 10). (I) Frequency of DCs and macrophages in the cecum, small intestine, and colon of naive WT mice ( n = 5). Each circle represents one mouse (B, C, and I) or one cell (E). Black line: median. Combined data of two (I), three (B), or nine (C) independent experiments. Statistical analysis: one-way ANOVA with Tukey’s correction (C) or Mann–Whitney U test (B). *, P ≤ 0.05; **, P ≤ 0.01.

    Article Snippet: TLR4 , BioRad , MCA2154T.

    Techniques: Activation Assay, Flow Cytometry, Injection, Expressing, Marker, MANN-WHITNEY

    TNF-mediated epithelial NF-κB activation occurs upon bacterial infection and induces an antibacterial response. (A) Representative two-photon microscopy images of cecal explants of streptomycin-pretreated p65 GFP-FL mice infected with S . Tm for 8–13.5 h ( n = 28). Categories for scoring of epithelial NF-κB activation status: “no activation” (green); “patchy activation” (blue); “full activation” (orange); “inflammation” (red; tissue distortion evident); “unspecified” (gray; was excluded from further analysis). Scale bars: 50 µm. (B) Distribution of the analyzed 28 samples of A among the four epithelial NF-κB activation categories (bottom). For simplification, the blue, orange, and red categories were summarized as “NF-κB signaling” (yellow, top). (C) Tnf transcript levels in the cecal mucosa of mice described in A and naive p65 GFP-FL mice, grouped according to the epithelial NF-κB activation status of the respective mice and depicted as 2 -ΔCT . Expression levels were normalized to Actb ( n = 33). (D) TNFa +/− or TNFa −/− > p65 GFP-FL x Tlr4 −/− BMCs were analyzed as described in A and B. (E) Log 2 ratios of selected genes in a transcriptome analysis of TNF-treated (8 h, 5 ng/ml) compared with untreated small intestinal epithelial organoids . FDR, false discovery rate. (F) C3 transcript levels in untreated and TNF-treated (5 ng/ml, 4 h) small intestinal organoids depicted as 2 -ΔCT . Expression levels were normalized to Actb . (G) Streptomycin-pretreated TNFa −/− mice and heterozygous littermates were orally infected with S . Tm for 36h. S . Tm in the cecal lumen (gated on O12 + cells, see ) were stained for surface C3 to assess coating of luminal bacteria by flow cytometry (C3 + population). MFI, median fluorescence intensity. Statistical analysis: Mann–Whitney U test (C, F, and G) or χ 2 test (D). *, P ≤ 0.05; **, P ≤ 0.01. Each circle represents one mouse (C and G) or one experiment (average; F). y axis in log 10 scale (C, F, and G). Combined data of three (G), four (F), five (A–C), or six (D) independent experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF

    doi: 10.1084/jem.20210862

    Figure Lengend Snippet: TNF-mediated epithelial NF-κB activation occurs upon bacterial infection and induces an antibacterial response. (A) Representative two-photon microscopy images of cecal explants of streptomycin-pretreated p65 GFP-FL mice infected with S . Tm for 8–13.5 h ( n = 28). Categories for scoring of epithelial NF-κB activation status: “no activation” (green); “patchy activation” (blue); “full activation” (orange); “inflammation” (red; tissue distortion evident); “unspecified” (gray; was excluded from further analysis). Scale bars: 50 µm. (B) Distribution of the analyzed 28 samples of A among the four epithelial NF-κB activation categories (bottom). For simplification, the blue, orange, and red categories were summarized as “NF-κB signaling” (yellow, top). (C) Tnf transcript levels in the cecal mucosa of mice described in A and naive p65 GFP-FL mice, grouped according to the epithelial NF-κB activation status of the respective mice and depicted as 2 -ΔCT . Expression levels were normalized to Actb ( n = 33). (D) TNFa +/− or TNFa −/− > p65 GFP-FL x Tlr4 −/− BMCs were analyzed as described in A and B. (E) Log 2 ratios of selected genes in a transcriptome analysis of TNF-treated (8 h, 5 ng/ml) compared with untreated small intestinal epithelial organoids . FDR, false discovery rate. (F) C3 transcript levels in untreated and TNF-treated (5 ng/ml, 4 h) small intestinal organoids depicted as 2 -ΔCT . Expression levels were normalized to Actb . (G) Streptomycin-pretreated TNFa −/− mice and heterozygous littermates were orally infected with S . Tm for 36h. S . Tm in the cecal lumen (gated on O12 + cells, see ) were stained for surface C3 to assess coating of luminal bacteria by flow cytometry (C3 + population). MFI, median fluorescence intensity. Statistical analysis: Mann–Whitney U test (C, F, and G) or χ 2 test (D). *, P ≤ 0.05; **, P ≤ 0.01. Each circle represents one mouse (C and G) or one experiment (average; F). y axis in log 10 scale (C, F, and G). Combined data of three (G), four (F), five (A–C), or six (D) independent experiments.

    Article Snippet: TLR4 , BioRad , MCA2154T.

    Techniques: Activation Assay, Infection, Microscopy, Expressing, Staining, Flow Cytometry, Fluorescence, MANN-WHITNEY

    Primary antibodies

    Journal: The Journal of Experimental Medicine

    Article Title: Intercrypt sentinel macrophages tune antibacterial NF-κB responses in gut epithelial cells via TNF

    doi: 10.1084/jem.20210862

    Figure Lengend Snippet: Primary antibodies

    Article Snippet: TLR4 , BioRad , MCA2154T.

    Techniques:

    Innate immune activation via TLR4/MD2.

    Journal: Molecules

    Article Title: Lipid A-Mediated Bacterial–Host Chemical Ecology: Synthetic Research of Bacterial Lipid As and Their Development as Adjuvants

    doi: 10.3390/molecules26206294

    Figure Lengend Snippet: Innate immune activation via TLR4/MD2.

    Article Snippet: In 2007, Ohto and Sato reported the crystal structure of the human MD-2 complex with lipid IVa ( 2 ) (antagonist) [ ], and Lee showed the crystal structure of the mouse TLR4/MD-2 complex with Eritoran ( 4 ) , a TLR4 antagonist developed by Eisai [ ].

    Techniques: Activation Assay

    Molecular mechanism of TLR4/MD2 dimerization process.

    Journal: Molecules

    Article Title: Lipid A-Mediated Bacterial–Host Chemical Ecology: Synthetic Research of Bacterial Lipid As and Their Development as Adjuvants

    doi: 10.3390/molecules26206294

    Figure Lengend Snippet: Molecular mechanism of TLR4/MD2 dimerization process.

    Article Snippet: In 2007, Ohto and Sato reported the crystal structure of the human MD-2 complex with lipid IVa ( 2 ) (antagonist) [ ], and Lee showed the crystal structure of the mouse TLR4/MD-2 complex with Eritoran ( 4 ) , a TLR4 antagonist developed by Eisai [ ].

    Techniques: