Journal: International Journal of Molecular Sciences

Article Title: Asprosin Exerts Pro-Inflammatory Effects in THP-1 Macrophages Mediated via the Toll-like Receptor 4 (TLR4) Pathway

doi: 10.3390/ijms24010227

Figure Lengend Snippet: Asprosin did not significantly affect Toll-like receptor 4 (TLR4) surface and intracellular expression. THP-1 macrophages were treated with either 10 nM asprosin, 100 nM asprosin or 100 ng/mL lipopolysaccharide (LPS). Cell surface expression of TLR4 was measured by flow cytometry after 4 and 24 h ( A ). Flow cytometry histograms display fluorescent intensity of the TLR4-PE stain ( x -axis) and cell count ( y -axis). Data were compared using one-way ANOVA and Dunnett’s multiple comparisons test (compared to control) ( n = 3). THP-1-cells (1 × 10 6 cells/well) were seeded and differentiated into macrophages in 6-well plates. Cells were then treated with either 100 nM asprosin or 100 ng/mL LPS for 4 and 24 h; collected and lysed for intracellular protein expression was measured by Western blotting ( B ). The presented Western data are representative of three independent experiments ( n = 3). CL: control; ASP: asprosin; LPS: lipopolysaccharide.

Article Snippet: To investigate the role of TLR4 in asprosin-induced inflammation, THP-1 derived macrophages were treated with 100 nM asprosin or 100 ng/mL LPS with and without a 1 h pre-treatment with 1 μM TAK-242 (R-Ethyl 6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-enecarboxylate), a TLR4 signalling inhibitor (#6587; Tocris Bioscience, Bristol, UK).

Techniques: Expressing, Flow Cytometry, Staining, Cell Counting, Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Asprosin Exerts Pro-Inflammatory Effects in THP-1 Macrophages Mediated via the Toll-like Receptor 4 (TLR4) Pathway

doi: 10.3390/ijms24010227

Figure Lengend Snippet: Asprosin-induced inflammation in THP-1 macrophages is inhibited by TAK-242, a Toll-like receptor 4 (TLR4) inhibitor. ( A ) Tumour necrosis factor α (TNFα); ( B ) Interleukin-1β (IL-1β); and ( C ) IL-8 mRNA levels were measured by qRT PCR in THP-1 macrophages treated with 1 μM TAK-242 for 1 h, prior to 4 h stimulation with 100 nM asprosin or 100 ng/mL lipopolysaccharide (LPS). Data are presented as fold-change (mean ± SEM) in transcript. The experiments were repeated in three independent cultures. One-way ANOVA; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Secreted levels of TNFα ( D ), IL-1β ( E ), MCP-1 ( F ) and IL-8 ( G ) were measured by a human cytokine BioPlex array in cell supernatant of THP-1 macrophages treated with 1μM TAK-242 for 1 h, prior to 4 h stimulation with 100 nM asprosin or 100 ng/mL LPS. The experiments were repeated in three independent cultures. One-way ANOVA; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: To investigate the role of TLR4 in asprosin-induced inflammation, THP-1 derived macrophages were treated with 100 nM asprosin or 100 ng/mL LPS with and without a 1 h pre-treatment with 1 μM TAK-242 (R-Ethyl 6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-enecarboxylate), a TLR4 signalling inhibitor (#6587; Tocris Bioscience, Bristol, UK).

Techniques: Quantitative RT-PCR

Journal: Nature Cancer

Article Title: Perivascular tenascin C triggers sequential activation of macrophages and endothelial cells to generate a pro-metastatic vascular niche in the lungs

doi: 10.1038/s43018-022-00353-6

Figure Lengend Snippet: a , Expression of INHBB , LAMA1 , SCGB3A1 , and OPG in ECs cocultured with control or TNC-stimulated macrophages. Expression was determined by qPCR. P values were calculated by paired one-tailed t test from 7 independent experiments. Boxes show median with upper and lower quartiles and whiskers indicate maximum and minimum values. b , Principal component analysis of bone marrow-derived macrophages treated with TNC or combination of TNC and TLR4i. c , GSEA showing enrichment of Toll-like receptor signaling pathway (KEGG) in macrophages treated with TNC or TNC+TLR4i. FDRs were determined from P values calculated by random gene set permutation test. d - g , Violin plot analyses of indicated gene signatures within the dataset of TNC-TLR4 stimulated macrophages. Gene signatures: metastasis-associated macrophages (up-regulated genes in metastasis-associated macrophages with log2FC> 2), M1 macrophages , M2 macrophages and wound healing . P values were determined by one-way ANOVA with Dunnett’s multiple comparison test from 3 biological replicates. h , Venn diagram showing number of genes of secreted proteins in bone marrow-derived macrophages, that are induced by TNC in a TLR4-dependent manner. i , Heatmap of genes induced by TNC and repressed by TLR4 inhibition. Selected examples are highlighted. j , Screen of 21 candidate factors to identify inducers of the four niche components. 20 candidates are from the list in panel i and one (NO, induced by diethylentriamine NONOate) was selected based on Nos2 upregulation by TNC (Fig. ). ST1.6R endothelial cells were stimulated with indicated factors and expression of INHBB , LAMA1 , SCGB3A1 and OPG analyzed by qPCR. *** P < 0.001, **** P < 0.0001 . P values were determined by one-way ANOVA with Dunnett’s multiple comparison test from minimum 4 independent experiments. Boxes show median with upper and lower quartiles and whiskers indicate maximum and minimum values. k , l , Violin plots analyzing expression of TNF response signature (SANA_TNF signaling, C2 in MSigDB) and NOS2 response signature (ZAMORA_NOS2 targets up, C2 in MSigDB) in ECs isolated from healthy mouse lungs or lungs harboring metastases. P values were calculated with an unpaired two-tailed t test from 3 biological replicates.

Article Snippet: For TLR4 inhibition, TAK-242 (Merck Millipore) was injected intraperitoneally at 10 mg kg –1 once per day.

Techniques: Expressing, Control, One-tailed Test, Derivative Assay, Comparison, Inhibition, Isolation, Two Tailed Test

Journal: Nature Cancer

Article Title: Perivascular tenascin C triggers sequential activation of macrophages and endothelial cells to generate a pro-metastatic vascular niche in the lungs

doi: 10.1038/s43018-022-00353-6

Figure Lengend Snippet: a , GSEA of GSP58, inflammation- or proliferation-related signatures expressed in ECs from mice with either macrophage-depleted lung metastases (clodronate-liposome) or metastases treated with anti-VEGF therapy (B20). b – d , Inhibition of TLR4 and VEGF in mice harboring lung metastasis. b , Experimental outline where mice were intravenously injected with indicated breast cancer cells and treated with TLR4i or B20 as single treatments, or together as a combination treatment. c , d , Bioluminescence analysis of metastatic colonization of lungs in mice injected with MDA231-LM2 ( c ) or 4T1 ( d ) cancer cells and treated as described in b . Left, representative bioluminescence images; right, quantification of metastatic lung colonization based on bioluminescence signal. MDA231-LM2, n = 11 mice; 4T1, n = 5 mice (single TLR4i or B20 treatments) and n = 4 mice (control or double treatment). P values were calculated by one-tailed Mann–Whitney test. e , Immunofluorescence analysis of apoptosis by expression of cleaved caspase 3 in metastatic nodules treated with TLR4i, B20 or a combination of the two; n = 5 mice for each group. Scale bar, 50 μm. P values were determined by one-tailed Mann–Whitney test. c – e , Boxes show median with upper and lower quartiles, and whiskers indicate maximum and minimum values. f , Model depicting two regulatory arms of vascular activation, where VEGF promotes proliferation of ECs and macrophages, stimulated by TNC–TLR4 signaling, promote inflammatory reaction in ECs and secretion of pro-metastatic factors of the vascular niche that are utilized by cancer cells. Shown are interactions between breast cancer cells and macrophages via the TNC–TLR4 axis, which lead to macrophage activation and subsequent induction of the perivascular niche by NO and TNF to produce pro-metastatic factors including INHBB, OPG, LAMA1 and SCGB3A1. INHBB and SCGB3A1 induce stem cell properties in breast cancer cells while OPG and LAMA1 promote survival of cancer cells at the metastatic site.

Article Snippet: For TLR4 inhibition, TAK-242 (Merck Millipore) was injected intraperitoneally at 10 mg kg –1 once per day.

Techniques: Inhibition, Injection, Control, One-tailed Test, MANN-WHITNEY, Immunofluorescence, Expressing, Activation Assay

Journal: Nature Cancer

Article Title: Perivascular tenascin C triggers sequential activation of macrophages and endothelial cells to generate a pro-metastatic vascular niche in the lungs

doi: 10.1038/s43018-022-00353-6

Figure Lengend Snippet: a , Immunofluorescence analysis of Ki67 expression in metastatic nodules after lung colonization by MDA231-LM2 breast cancer cells followed by treatment with TLR4i, anti-VEGF (B20) or combination of the two. Left, representative fluorescence images. Right, quantification; n = 5 mice for each group. Boxes show median with upper and lower quartiles and whiskers indicate maximum and minimum values. P values were determined by one-tailed Mann-Whitney test. Scale bar, 50 μm. b , c , Metastasis analysis in an orthotopic metastasis model where mice were treated with TLR4i, B20 or both, after the primary tumors had been surgically removed. Shown are experimental procedures (b) and quantification of metastasis (c) with representative examples of bioluminescence in lungs (left) and normalized photon flux (right); Control n = 13 mice; TLR4i n = 13 mice; B20 n = 12 mice; TLR4i + B20 n = 12 mice. Data are means +/- s.e.m. P value was calculated by one-tailed Mann-Whitney test. d , Kaplan Meier analysis showing overall survival of breast cancer patients (METABRIC discovery) stratified according to expression of VEGF signature (VEGF-S) and TLR4 signature (TLR4-S) . Survival analysis spanning 10 years is shown. Patients were divided based on the median value of each signatures; Patient groups: VEGF-S/TLR4-S High/High n = 239; High/Low n = 259; Low/High n = 264; Low/Low n = 233. Shown is hazard ratio (HR) for the comparison VEGF-S/TLR4-S low/low vs high/high. P value was determined by Log-rank test.

Article Snippet: For TLR4 inhibition, TAK-242 (Merck Millipore) was injected intraperitoneally at 10 mg kg –1 once per day.

Techniques: Immunofluorescence, Expressing, Fluorescence, One-tailed Test, MANN-WHITNEY, Control, Comparison