anti il 1β neutralizing antibodies  (R&D Systems)

Journal: Cancer Communications

Article Title: The Ly6g high Neutrophil Subset Dictates Breast Cancer Lung Metastasis via CD8 + T Cell Death

doi: 10.34133/cancomm.0003

Figure Lengend Snippet: Il-1β induces Ly6g high neutrophil NETosis in the lung metastatic niche. (A) Heatmap of the scRNA-seq data showing the expression of cytokine genes at different time points during lung metastasis. (B and C) Representative immunofluorescence micrographs (B) showing NET formation by FACS-sorted Ly6g high and Ly6g low neutrophils ( n = 6) after treatment with Il-1β, Cxcl2, and Ccl6 for 6 h in vitro. NETs were stained with antibodies against Mpo (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). The statistical data are presented in (C). (D) Representative immunofluorescence micrographs showing NET formation at the MACRO stages of lung tissue with PBS, rIl-1β, anti-IgG, and anti-Il-1β antibody treatment, respectively [4T1-LM3 (BALB/c) model, n = 5]. NETs were stained with antibodies against Mpo (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). The bar graph on the right quantifies NET formation. (E) Representative bioluminescence imaging and hematoxylin and eosin (H&E) staining images at the MACRO lungs from mice treated with PBS, rIl-1β, IgG, or anti-Il-1β antibody [4T1-LM3 (BALB/c) model, n = 5]. The bar graph on the right shows the quantitative data of lung metastasis burden. (F) Violin plots showing the expression of Il1b in different cell clusters in the lung tissues based on scRNA-seq data from Fig. D. (G) Representative immunofluorescence micrographs demonstrate NET formation in sorted Ly6g high neutrophils ( n = 6). Neutrophils were treated with CM-MΦ or CM-MΦ that had been neutralized with an anti-Il-1β antibody. NETs were stained with antibodies against Mpo (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). (H and I) Mice were treated with anti-IgG control, anti-F4/80 antibody, or anti-F4/80 antibody combined with rIl-1β until the macrometastatic stage [4T1-LM3 (BALB/c) model, n = 6]. (H) Il-1β levels in the lungs were detected by ELISA. (I) Representative immunofluorescence images show NET formation. NETs were stained for Mpo (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). The bar graph on the right quantifies NET formation (I). (J) Macrophages were treated with CM-Neu, NETs (5 μg/ml), NETs (10 μg/ml), or NETs (10 μg/ml) combined with deoxyribonuclease (DNase) I ( n = 3). The expression of Il1b was determined by qPCR. The data with error bars are presented as the mean ± SD; statistical significance was determined by 2-way ANOVA (C) and 1-way ANOVA test (D, E, and G to J). 4T1-LM3, 4T1-lung metastasis 3; ANOVA, analysis of variance; Ccl11 , c-c motif chemokine ligand 11; Ccl12 , c-c motif chemokine ligand 12; Ccl17 , c-c motif chemokine ligand 17; Ccl2 , c-c motif chemokine ligand 2; Ccl22 , c-c motif chemokine ligand 22; Ccl3 , c-c motif chemokine ligand 3; Ccl4 , c-c motif chemokine ligand 4; Ccl5 , c-c motif chemokine ligand 5; Ccl6 , c-c motif chemokine ligand 6; CCL6; c-c motif ligand 6; Ccl9 , c-c motif chemokine ligand 9; CM-MΦ, macrophage-derived conditioned medium; CM-Neu, neutrophil-derived conditioned medium; Cxcl12 , c-x-c motif chemokine ligand 12; Cxcl14 , c-x-c motif chemokine ligand 14; Cxcl16 , c-x-c motif chemokine ligand 16; CXCL2, c-x-c motif chemokine ligand 2; Cxcl2 , c-x-c motif chemokine ligand 2; Cxcl3 , c-x-c motif chemokine ligand 3; Cxcl9 , c-x-c motif chemokine ligand 9; DAPI, 4’,6-diamidino-2-phenylindole; ELISA, enzyme linked immunosorbent assay; FACS, fluorescence-activated cell sorting; H3cit; citrullinated histone H3; Il12a , interleukin 12a; Il13 , interleukin 13; Il18 , interleukin, 18; Il1a , interleukin 1α; Il1b , interleukin 1β; Il-1β, interleukin-1β; Il2 ,interleukin 2; Il33 , interleukin 33; Il4 , interleukin 4; Il6 , interleukin 6; Ly6g, lymphocyte antigen 6 complex locus g; MACRO, macrometastatic lung; MICRO, micrometastatic lung; MPO, myeloperoxidase; NETs, neutrophil extracellular trap; NK, natural killer; NL, normal lung; Ppbp , pro-platelet basic protein; Neu, neutrophil; PRE, premetastatic lung; qRT-PCR, quantitative real-time polymerase chain reaction; rIl-1β, recombinant interleukin-1β; scRNA-seq: single-cell RNA sequencing; SD, standard deviation.

Article Snippet: We then treated Ly6g high and Ly6g low neutrophils with this CM-MΦ, in the presence or absence of an Il-1β neutralizing antibody (5 μg/ml, BE0246, BioXCell), for 24 h. After treatment, the cells were incubated with the ROS-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; 10 μM, 50101ES01, Yeasen) at 37 °C for 20 min, washed twice with PBS, and analyzed immediately using a BD FACSCanto Plus flow cytometer.

Techniques: Expressing, Immunofluorescence, In Vitro, Staining, Imaging, Control, Enzyme-linked Immunosorbent Assay, Derivative Assay, Fluorescence, FACS, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Recombinant, RNA Sequencing, Standard Deviation

Journal: Cancer Communications

Article Title: The Ly6g high Neutrophil Subset Dictates Breast Cancer Lung Metastasis via CD8 + T Cell Death

doi: 10.34133/cancomm.0003

Figure Lengend Snippet: Prognostic significance of NETs in human BC. (A) Representative FACS plot showing the ratio of human CD84 high and CD84 low neutrophils in healthy individuals ( n = 50) and patients with BC at different stages [stages I/II ( n = 80), stages III/IV ( n = 80)]. To define the CD84 high and CD84 low subsets in humans, we first established the positive gating threshold using FMO controls. Subsequently, the boundary between “high” and “low” subsets was determined based on a clear inflection point observed in the fluorescence intensity histogram. Statistical significance was determined by comparing with the healthy group. The bar graph on the right quantifies the ratio of human CD84 high and CD84 low neutrophils. (B) Representative immunofluorescence micrographs showing NET formation of CD84 high and CD84 low neutrophils, which were sorted by FACS after treatment with PMA for 2 h ( n = 6). NETs were stained with antibodies against MPO (red) and H3cit (green), and nuclei were counterstained with DAPI (blue). The bar graph on the right quantifies the formation of NETs. (C) Plasma NET levels in healthy individuals ( n = 50) and BC patients at different stages [stages I/II ( n = 80), stages III/IV ( n = 80)]. (D) Kaplan–Meier survival curves showing the overall survival (OS) of BC patients with low (NETs < 344.91 pg/ml; n = 83) or high (NETs ≥ 344.91 pg/ml; n = 77) concentrations of plasma NETs. BC patients were stratified into high and low NET groups using the mean plasma NET level of the entire cohort as the cutoff. (E) Receiver operator characteristic (ROC) curve analysis of plasma NET levels for predicting BC patients’ lung metastases ( n = 160). The area under the curve (AUC) value reflects the model’s power to distinguish between BC patients with and without lung metastasis within 6 years after diagnosis. Higher AUC values (approaching 1) denote superior differentiation accuracy at this time point. (F) Correlation between plasma NET levels and CD8 + T cell proportion in healthy individuals and patients with BC ( n = 210). (G) Kaplan–Meier analysis showing the recurrence-free survival of BC patients with high or low levels of LL37 ( n = 4,929). Data were obtained from the Kaplan–Meier plotter database, which does not provide detailed numerical thresholds for LL37 level classification. (H) Mechanism scheme of Ly6g high and Ly6g low neutrophils in promoting pulmonary metastasis of BC. Briefly, Ly6g high neutrophils accumulated in the premetastatic stage and induced CD8 + T cell apoptosis through NETosis. The NET-derived cathelicidin directly bound with Ant1, an mPTP protein in CD8 + T cells, leading to conformational changes in the Ant1 and subsequent Ant1–Vdac1 complex formation, which resulted in mPTP opening, loss of ΔΨm, and uncoupling of mitochondrial electron transport chain in CD8 + T cells. Ly6g low neutrophils bearing MDSC-like transcriptional signatures exhibit a superior capacity to inhibit the proliferation and effector functions of CD8 + T cells. The data with error bars are presented as the mean ± SD; statistical significance was determined by 2-way ANOVA (A), Student’s t test (B), 1-way ANOVA test (C), and 2-sided log-rank test (D and G). 4T1-LM3, 4T1-lung metastasis 3; ANOVA, analysis of variance; APC, allophycocyanin; BC, breast cancer; CD8, cluster of differentiation 8; CD84, cluster of differentiation 84; CI, confidence interval; DAPI, 4',6-diamidino-2-phenylindole; E0771-LM3, E0771-lung metastasis 3; FACS, fluorescence-activated cell sorting; FMO, fluorescence-minus-one; H3cit, citrullinated histone H3; HR, hazard ratio; Interferon-γ, IFN-γ; Il-1β, interleukin-1β; Ly6g, lymphocyte antigen 6 complex locus g; MDSC, myeloid-derived suppressor cell; MPO, myeloperoxidase; mPTP, mitochondrial permeability transition pore; NETs, neutrophil extracellular traps; PADI4, peptidyl arginine deiminase 4; PE, phycoerythrin; PMA, phorbol 12-myristate 13-acetate; RFS, recurrence-free survival; ROS, reactive oxygen species; Vdac1, voltage-dependent anion channel 1; SD, standard deviation; ΔΨm, mitochondrial membrane potential.

Article Snippet: We then treated Ly6g high and Ly6g low neutrophils with this CM-MΦ, in the presence or absence of an Il-1β neutralizing antibody (5 μg/ml, BE0246, BioXCell), for 24 h. After treatment, the cells were incubated with the ROS-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA; 10 μM, 50101ES01, Yeasen) at 37 °C for 20 min, washed twice with PBS, and analyzed immediately using a BD FACSCanto Plus flow cytometer.

Techniques: Fluorescence, Immunofluorescence, Staining, Clinical Proteomics, Biomarker Discovery, Derivative Assay, FACS, Permeability, Standard Deviation, Membrane

Journal: Science advances

Article Title: The P2X7R/NLRP3 inflammasome axis suppresses enthesis regeneration through inflammatory and metabolic macrophage-stem cell cross-talk.

doi: 10.1126/sciadv.adr4894

Figure Lengend Snippet: Fig. 1. NLRP3 inflammasomes are activated in macrophages at the injured enthesis. (A) Schematic diagram of RNA-seq and validation of DEGs. (B) PCA of the sham operation and RCTR groups. (C) Heat map of DEGs between the sham operation and RCTR groups. (D) Relative mRNA expression levels of Il1b, Caspase-1, Nlrp3, and P2rx7 in the enthesis of RCTR groups in comparison to sham operation groups. (E) Immunofluorescence (IF) staining of CD68 (red), NLRP3 (green), and caspase-1 (magenta) in the injured enthesis of wild-type and Nlrp3−/− mice at 3 dpi. Orange and green dashed squares represent enlarged images of the enthesis. Arrows indicate specks of NLRP3 and caspase-1. (F) Quantification of specks per macrophage in the injured enthesis of wild-type and Nlrp3−/− mice at 3 dpi. (G and H) The relative caspase-1 activity and IL-1β concentration of the enthesis in wild-type and Nlrp3−/− mice at 3, 7, 14, and 28 dpi. T, tendon; I, tendon-to-bone interface; B, bone. Data are presented as means ± SD. Statistical significance was determined using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. d, days.

Article Snippet: For antibodies treatment experiments, 200 μg of IL- 1β neutralizing antibodies (BioXcell, New Hampshire, USA, BE0246) or control IgG (BioXcell, New Hampshire, USA, BE0091) was injected into the joint cavity near the injured enthesis at 3 and 7 dpi.

Techniques: RNA Sequencing, Biomarker Discovery, Expressing, Comparison, Immunofluorescence, Staining, Activity Assay, Concentration Assay

Journal: Science advances

Article Title: The P2X7R/NLRP3 inflammasome axis suppresses enthesis regeneration through inflammatory and metabolic macrophage-stem cell cross-talk.

doi: 10.1126/sciadv.adr4894

Figure Lengend Snippet: Fig. 3. scRNA-seq uncovers that NLRP3 inflammasomes deteriorate inflammation and IL-1β inflammatory cross-talk. (A) Schematic of scRNA-seq, in which the enthesis was harvested from wild-type controls and Nlrp3−/− mice at 7 dpi and processed for scRNA-seq. (B) UMAP plot of 56,217 cells from wild-type controls (n = 3) and Nlrp3−/− mice (n = 3). (C) Bar plot of the proportions of nine major cell clusters in the injured enthesis of wild-type controls and Nlrp3−/− mice at 7 dpi. (D) Violin plots of specific gene expressions in macrophages, mesenchymal cells, and neutrophils. (E) GO enrichment analysis of down-regulated genes in PIM and up-regulated genes in AIM in Nlrp3−/− mice. (F) Circle plot of the interactions of subsets of macrophages and mesenchymal cells. Edge line thickness suggests the interaction strength between different cell clusters. (G) NicheNet analysis of ligand-target regulatory potential between macrophages and mesenchymal stem cells. (H) Gene set cores of IL-1 signaling in different mesenchymal cell subsets in wild-type controls and Nlrp3−/− mice. Statistical significance was determined using Student’s t test. FACS, fluorescence-activated cell sorting; TGF-β, transforming growth factor–β.

Article Snippet: For antibodies treatment experiments, 200 μg of IL- 1β neutralizing antibodies (BioXcell, New Hampshire, USA, BE0246) or control IgG (BioXcell, New Hampshire, USA, BE0091) was injected into the joint cavity near the injured enthesis at 3 and 7 dpi.

Techniques: Fluorescence, FACS

Journal: Science advances

Article Title: The P2X7R/NLRP3 inflammasome axis suppresses enthesis regeneration through inflammatory and metabolic macrophage-stem cell cross-talk.

doi: 10.1126/sciadv.adr4894

Figure Lengend Snippet: Fig. 4. Blocking IL-1β inflammatory cross-talk with neutralizing antibodies accelerates enthesis regeneration. (A) Schematic of animal experiments, in which the RCTR model was established, and IL-1β neutralizing antibodies or control IgG was injected into the articular cavity, with analyses at 14 and 28 dpi. (B) H&E and toluidine blue staining of the enthesis in mice with IL-1β neutralizing antibodies or control IgG injection at 14 and 28 dpi. Black dashed squares represent the enlarged images of the enthesis. (C and D) Histological scores and metachromasia area size of the enthesis in mice with IL-1β neutralizing antibodies or control IgG injection at 14 and 28 dpi. (E) Micro-CT coronal views of the humerus in mice with IL-1β neutralizing antibodies or control IgG injection at 14 and 28 dpi. Green dashed squares represent the area of the enthesis. (F to H) Quantitative analysis of BMD, BV/TV, and Tb. N of the enthesis in mice with IL-1β neutralizing antibodies or control IgG injection and sham operation. (I) Deformation and load curves of the enthesis in mice with IL-1β neutralizing antibodies or control IgG injection at 14 and 28 dpi. (J to L) Failure load, stiffness, and work of the enthesis in mice with IL-1β neutralizing antibodies or control IgG injection at 14 and 28 dpi. Data are presented as means ± SD. Statistical significance was deter- mined using one-way ANOVA with Tukey’s multiple comparisons test and Student’s t test.

Article Snippet: For antibodies treatment experiments, 200 μg of IL- 1β neutralizing antibodies (BioXcell, New Hampshire, USA, BE0246) or control IgG (BioXcell, New Hampshire, USA, BE0091) was injected into the joint cavity near the injured enthesis at 3 and 7 dpi.

Techniques: Blocking Assay, Control, Injection, Staining, Micro-CT

Journal: Science advances

Article Title: The P2X7R/NLRP3 inflammasome axis suppresses enthesis regeneration through inflammatory and metabolic macrophage-stem cell cross-talk.

doi: 10.1126/sciadv.adr4894

Figure Lengend Snippet: Fig. 5. NLRP3 inflammasomes suppress the secretion of anti-inflammatory cytokines by macrophages to inhibit inflammation resolution. (A) IL-1β concentration in the supernatant of wild-type and Nlrp3−/− BMDMs. (B) Scan images of the supernatant of wild-type and Nlrp3−/− BMDMs in the mouse inflammation array Q1. (C) Heat- map of inflammation factors in the supernatant of wild-type and Nlrp3−/− BMDMs. (D) Concentrations of IL-1β, IFN-γ, IL-6, TNF-α, IL-1α, IL-10, IL-13, and IL-4 in the super- natant of wild-type and Nlrp3−/− BMDMs. (E to H) IHC staining and ELISA analysis of IL-10 and IL-13 in the enthesis of wild-type and Nlrp3−/− mice. Green dashed squares represent the enlarged images of the enthesis. (I and J) Immunofluorescence staining and quantification of CD68 (green)– and CD206 (red)–positive cells in the enthesis of wild-type controls and Nlrp3−/− mice. Red dashed squares represent the enlarged images of the enthesis. Arrows indicate CD68+ and CD206+ macrophages. Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test and Student’s t test.

Article Snippet: For antibodies treatment experiments, 200 μg of IL- 1β neutralizing antibodies (BioXcell, New Hampshire, USA, BE0246) or control IgG (BioXcell, New Hampshire, USA, BE0091) was injected into the joint cavity near the injured enthesis at 3 and 7 dpi.

Techniques: Concentration Assay, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Science advances

Article Title: The P2X7R/NLRP3 inflammasome axis suppresses enthesis regeneration through inflammatory and metabolic macrophage-stem cell cross-talk.

doi: 10.1126/sciadv.adr4894

Figure Lengend Snippet: Fig. 8. Conditional KO of P2rx7 in myeloid cells reduces NLRP3 inflammasome activation after enthesis injury and improves enthesis regeneration. (A) Immuno- fluorescence staining of CD68 (green) and P2X7R (yellow) in native and injured enthesis. Red dashed squares represent enlarged images of the enthesis. (B) Feature plots of single-cell gene expression of P2rx7 in macrophages in wild-type mice. (C and D) The relative caspase-1 activity and concentration of IL-1β in the enthesis of Lyz2-P2rx7f/f and Lyz2-P2rx7−/− mice at 3, 7, 14, and 28 dpi. (E) Schematic of animal experiments, in which the RCTR model was established in Lyz2-P2rx7f/f and Lyz2-P2rx7−/− mice, and analyzed at 3, 7, 14, and 28 dpi. (F) H&E and toluidine blue staining of the enthesis in Lyz2-P2rx7f/f and Lyz2-P2rx7−/− mice at 14 and 28 dpi. Black dashed squares represent enlarged images of the enthesis. (G and H) Histological scores and the metachromasia area size of the enthesis in Lyz2-P2rx7f/f and Lyz2-P2rx7−/− mice at 14 and 28 dpi. (I) Micro-CT coronal views of the humerus of Lyz2-P2rx7f/f and Lyz2-P2rx7−/− mice at 14 and 28 dpi. Green dashed squares represent the area of interest. (J and K) Quantitative analysis of the BMD and BV/TV of the enthesis. (L) Deformation and load curves of the enthesis in Lyz2-P2rx7 f/f and Lyz2-P2rx7−/− mice at 14 and 28 dpi. (M to O) Failure load, stiffness, and work of the enthesis in mice with IL-1β neutralizing antibodies or control IgG injection at 14 and 28 dpi. Data are presented as means ± SD. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test and Student’s t test.

Article Snippet: For antibodies treatment experiments, 200 μg of IL- 1β neutralizing antibodies (BioXcell, New Hampshire, USA, BE0246) or control IgG (BioXcell, New Hampshire, USA, BE0091) was injected into the joint cavity near the injured enthesis at 3 and 7 dpi.

Techniques: Activation Assay, Fluorescence, Staining, Gene Expression, Activity Assay, Concentration Assay, Micro-CT, Control, Injection

Journal: Science advances

Article Title: The P2X7R/NLRP3 inflammasome axis suppresses enthesis regeneration through inflammatory and metabolic macrophage-stem cell cross-talk.

doi: 10.1126/sciadv.adr4894

Figure Lengend Snippet: Fig. 9. The schematic of this study. After enthesis injury, NLRP3 inflammasomes are activated in infiltrated macrophages upon receiving activation signals mediated by P2X7R. The activation of the P2X7R/NLRP3 inflammasome axis not only exacerbates inflammation by prompting the release of IL-1β and suppressing the production of anti-inflammatory factors including IL-10 and IL-13 but also inhibits the production of proregenerative docosatrienoic acid. NLRP3 inflammasomes suppress enthesis re- generation via aggravating IL-1β inflammatory cross-talk and restraining docosatrienoic acid metabolic cross-talk between macrophages and stem cells. Blocking the P2X7R/NLRP3 inflammasome axis rewires the cross-talk between macrophages and stem cells and converts pathological inflammation to reparative inflammation. This study illustrates that the P2X7R/NLRP3 inflammasome axis is a promising regenerative therapeutic target for enthesis injury treatment.

Article Snippet: For antibodies treatment experiments, 200 μg of IL- 1β neutralizing antibodies (BioXcell, New Hampshire, USA, BE0246) or control IgG (BioXcell, New Hampshire, USA, BE0091) was injected into the joint cavity near the injured enthesis at 3 and 7 dpi.

Techniques: Activation Assay, Blocking Assay

Journal: Scientific Reports

Article Title: A pivotal role for the IL-1β and the inflammasome in preterm labor

doi: 10.1038/s41598-024-54507-w

Figure Lengend Snippet: IL-1β is over-expressed in myometrium during labor. ( a ) Pictures of myometrium from non-laboring and laboring patients during normal pregnancy (NP) or with chorioamnionitis (CA). Images of IL-1β or hemalum eosine staining are representative of 5 pictures taken from 3 different samples for each condition with an Axiozoom epifluorescence microscope (×400) in random chosen fields. ( b ) Fluorescence intensity of IL-1β labeling, mean ± SEM, n = 15, **P < 0.01 and ****P < 0.0001.

Article Snippet: IL-1β neutralizing antibody (αIL-1β, mabg-hIL-1b-3) was obtained from Invivogen (San Diego, USA) and human IL-1β Quantikine ELISA KIT from R&D Systems (Abingdon, UK).

Techniques: Staining, Microscopy, Fluorescence, Labeling

Journal: Scientific Reports

Article Title: A pivotal role for the IL-1β and the inflammasome in preterm labor

doi: 10.1038/s41598-024-54507-w

Figure Lengend Snippet: IL-1β is required for contractions of collagen lattices of primary human myometrial cells co-cultured with macrophages. ( a ) Schematic representation of the LPS-mediated IL-1β effects on myometrial cells contraction cultured with macrophages. ( b ) IL-1β secretion in co-culture cell medium. Macrophages and myometrial cells were cultured for 24 h in presence or absence of LPS (100 ng/ml). Supernatants were collected and IL-1β was evaluated with ELISA assay. Concentrations were standardized with protein cell content and presented as means ± SEM, n = 6, **P < 0.01. ( c ) Representative photographs of collagen lattices of myometrial cells co-cultured with macrophages stimulated with LPS (100 ng/ml), in presence or absence of Anakinra (10 µg/ml) or αIL-1β (1 µg/ml) at 96 h. ( d ) Mean percentage contraction of collagen lattices after 96 h of stimulation with LPS or LPS + Anakinra (10 µg/ml) or LPS + αIL-1β (1 µg/ml) compared to the basal surface area, mean ± SEM, n = 5, *P < 0.05, **P < 0.01 and ****P < 0.0001. ( e ) Schematic representation of the IL-1β effects on myometrial cells contraction. f) Photographs of collagen lattices of myometrial cells alone in presence of LPS (100 ng/ml) or IL-1β (1 ng/ml). ( g ) Mean percentage contraction of collagen lattices ± SEM after 96 h of LPS or IL-1β treatment, compared to the basal surface area.

Article Snippet: IL-1β neutralizing antibody (αIL-1β, mabg-hIL-1b-3) was obtained from Invivogen (San Diego, USA) and human IL-1β Quantikine ELISA KIT from R&D Systems (Abingdon, UK).

Techniques: Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: A pivotal role for the IL-1β and the inflammasome in preterm labor

doi: 10.1038/s41598-024-54507-w

Figure Lengend Snippet: IL-1β has different effects on the differentiation of primary myometrial smooth muscle cells. ( a ) Schematic representation of IL-1β effects on differentiation of myometrial cells. ( b ) Myometrial cells were treated with LPS (100 ng/ml) or IL-1β (1 ng/ml) and stained with phalloidin (green), vinculin (purple) and DAPI (blue) for nuclear localization. Images, taken with an Axiozoom epifluorescence microscope (×400), are representative of 5 random pictures of four independent experiments. ( c ) Mean fluorescence intensity of phalloidin or vinculin ± SEM, n = 20, **P < 0.01, ***P < 0.001 and ****P < 0.0001. ( d ) Fluorescence images of LY (green) and RD (red) transfer to adjacent cells after scrap loading. Cells were treated for 24 h with LPS or IL-1β. Pictures were taken with an inverted microscope at ×100 magnification in two random chosen fields and are representative of three experiments. ( e ) Mean surface of LY staining ± SEM, n = 6.

Article Snippet: IL-1β neutralizing antibody (αIL-1β, mabg-hIL-1b-3) was obtained from Invivogen (San Diego, USA) and human IL-1β Quantikine ELISA KIT from R&D Systems (Abingdon, UK).

Techniques: Staining, Microscopy, Fluorescence, Inverted Microscopy

Journal: Scientific Reports

Article Title: A pivotal role for the IL-1β and the inflammasome in preterm labor

doi: 10.1038/s41598-024-54507-w

Figure Lengend Snippet: Neutralizing IL-1β effect prevents the differentiation of primary human myometrial smooth muscle cells. ( a ) Schematic representation of the LPS effects on differentiation of myometrial cells in a co-culture model. ( b ) Representative images of myometrial cells and macrophages co-cultures treated with LPS (100 ng/ml) in presence or absence of Anakinra (1 µg/ml) or αIL-1β (100 ng/ml) and stained with phalloidin (green), vinculin (purple) and DAPI (blue) for nuclear localization. Images, taken with an Axiozoom epifluorescence microscope (×400), are representative of 5 random pictures of four independent experiments. ( c ) Mean fluorescence intensity of phalloidin or vinculin ± SEM, n = 20, **P < 0.01, ***P < 0.001, ****P < 0.0001. ( d ) Fluorescence images of LY (green) and RD (red) transfer to adjacent cells after scrap loading. Cells were treated for 24 h with LPS ± Anakinra or αIL-1β. Pictures were taken with an inverted microscope at ×100 magnification in two random chosen fields and are representative of three experiments. ( e ) Mean surface of LY staining ± SEM, n = 6, ****P < 0.0001.

Article Snippet: IL-1β neutralizing antibody (αIL-1β, mabg-hIL-1b-3) was obtained from Invivogen (San Diego, USA) and human IL-1β Quantikine ELISA KIT from R&D Systems (Abingdon, UK).

Techniques: Co-Culture Assay, Staining, Microscopy, Fluorescence, Inverted Microscopy

Journal: Scientific Reports

Article Title: A pivotal role for the IL-1β and the inflammasome in preterm labor

doi: 10.1038/s41598-024-54507-w

Figure Lengend Snippet: Macrophages play a pivotal role in LPS-induced differentiation by secreting IL-1β. ( a ) IL-1β concentration in macrophages or myometrial cells stimulation supernatants. Macrophages or myocytes were cultured for 24 h in presence or absence of LPS (100 ng/ml). Supernatants were collected and IL-1β concentrations were evaluated with ELISA assay and standardized with protein cell content ± SEM, n = 6, *** P < 0.001. ( b ) Schematic representation of the effects of IL-1β blockage for each cell type on differentiation of myometrial cells in a Transwell assay. ( c ) Transwell experiments with macrophages (upper compartment) and myometrial cells (lower compartment) following treatment with LPS (100 ng/ml) in presence or absence of Anakinra (1 µg/ml) or αIL-1β (100 ng/ml). Representative images of phalloidin (green), vinculin (purple) and DAPI for nuclear localization (blue) were taken with an Axiozoom epifluorescence microscope (×400) in five random chosen fields of four separated experiments. ( d ) Mean fluorescence intensity of phalloidin or vinculin ± SEM, n = 20, ****P < 0.0001. ( e ) Fluorescence images of LY (green) and RD (red) transfer to adjacent cells after scrap loading. Cells were treated for 24 h with LPS in the upper compartment and with Anakinra or αIL-1β in the lower compartment. Pictures were taken with an inverted microscope at ×100 magnification in two random chosen fields and are representative of three experiments. ( f ) Mean surface of LY staining ± SEM, n = 6, ****P < 0.0001.

Article Snippet: IL-1β neutralizing antibody (αIL-1β, mabg-hIL-1b-3) was obtained from Invivogen (San Diego, USA) and human IL-1β Quantikine ELISA KIT from R&D Systems (Abingdon, UK).

Techniques: Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Transwell Assay, Microscopy, Fluorescence, Inverted Microscopy, Staining

Journal: Scientific Reports

Article Title: A pivotal role for the IL-1β and the inflammasome in preterm labor

doi: 10.1038/s41598-024-54507-w

Figure Lengend Snippet: Inhibition of NLRP3 inflammasome prevents LPS-induced contractions of collagen lattices of primary human myometrial cells co-cultured with macrophages. ( a ) Schematic representation of the effects of NLRP3 inflammasome inhibition on differentiation of myometrial cells in a co-culture model. ( b ) Macrophages and myometrial cells were co-cultured for 24 h in presence or absence of LPS (100 ng/ml) ± MCC950 (0.1 µM). Supernatants were collected and IL-1β concentrations were evaluated with ELISA assay and standardized with protein cell content ± SEM, n = 6, *P < 0.05 and **P < 0.01. ( c ) Representative photographs of collagen lattices of myometrial cells co-cultured with macrophages stimulated with LPS (100 ng/ml) ± MCC950 for 96 h. ( d ) Mean percentage contraction of collagen lattices after 96 h of stimulation with LPS in presence or absence of MCC950 compared to the basal surface area, mean ± SEM, n = 5, *P < 0.05.

Article Snippet: IL-1β neutralizing antibody (αIL-1β, mabg-hIL-1b-3) was obtained from Invivogen (San Diego, USA) and human IL-1β Quantikine ELISA KIT from R&D Systems (Abingdon, UK).

Techniques: Inhibition, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: A pivotal role for the IL-1β and the inflammasome in preterm labor

doi: 10.1038/s41598-024-54507-w

Figure Lengend Snippet: Inhibition of the macrophages’ inflammasome blocks LPS-induced myometrial synchronization. ( a ) Fluorescence images of LY (green) and RD (red) transfer to adjacent cells after scrap loading. Cells were treated for 24 h with LPS in the upper compartment and with Anakinra or αIL-1β in the lower compartment. Pictures were taken with an inverted microscope at ×100 magnification in two random chosen fields and are representative of three experiments. ( b ) Mean surface of LY staining ± SEM, n = 6, ****P < 0.0001.

Article Snippet: IL-1β neutralizing antibody (αIL-1β, mabg-hIL-1b-3) was obtained from Invivogen (San Diego, USA) and human IL-1β Quantikine ELISA KIT from R&D Systems (Abingdon, UK).

Techniques: Inhibition, Fluorescence, Inverted Microscopy, Staining

Journal: Scientific Reports

Article Title: A pivotal role for the IL-1β and the inflammasome in preterm labor

doi: 10.1038/s41598-024-54507-w

Figure Lengend Snippet: Schematic representation of putative interactions between myometrial smooth muscle cells and macrophages. This representation was a synthesis of the data presented in this article and previous publications from our team: Hadi et al . and Wendremaire et al. . LPS treatment of myometrial and macrophages co-culture leads to the production by macrophages of ROS which contributed to inflammasome activation and IL-1β processing. Myometrial cytoskeleton reorganization and synchronization is directly achieved by IL-1β and ROS.

Article Snippet: IL-1β neutralizing antibody (αIL-1β, mabg-hIL-1b-3) was obtained from Invivogen (San Diego, USA) and human IL-1β Quantikine ELISA KIT from R&D Systems (Abingdon, UK).

Techniques: Co-Culture Assay, Activation Assay