anti tl1a  (R&D Systems)

Journal: Respiratory research

Article Title: Necroptosis plays a role in TL1A-induced airway inflammation and barrier damage in asthma.

doi: 10.1186/s12931-024-02900-4

Figure Lengend Snippet: Fig. 4 Administration of TL1A directly induces features of necroptosis. (A) HBE cells were treated with BV6 (0.5 µM) and zVAD-fmk (20 µM) for 2 h, followed by stimulation with TL1A for 18 h. Viability was determined by CCK8 (n = 3). (B) The dosing regimen of recombinant protein TL1A. (C) Total number of cells and total protein content in BALF (n = 4). (D) Western blot analysis of p-RIPK3, t-RIPK3, p-MLKL, and t-MLKL in lungs of mice (n = 3). (E) Representative photomicrographs for lung paraffin sections from mice stained with H&E, PAS, and Masson staining, along with quantification of inflammation scores, PAS-positive cells, and collagen deposition around the airways (n = 5–8). Bars = 50 μm.*p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The methods of asthma model construction, RIPK3 inhibitor GSK872 (Selleck, China) injection, and recombinant TL1A (R&D systems, USA) nasal administration were illustrated in the figures below.

Techniques: Recombinant, Western Blot, Staining

Journal: Respiratory research

Article Title: Necroptosis plays a role in TL1A-induced airway inflammation and barrier damage in asthma.

doi: 10.1186/s12931-024-02900-4

Figure Lengend Snippet: Fig. 5 TL1A-induced necroptosis promotes asthma inflammation. (A) OVA-induced asthma mouse model experimental scheme and recombinant pro tein TL1A dosing schedule. (B) Total number of cells and total protein content in BALF (n = 4). (C) Western blot analysis of p-RIPK3, t-RIPK3, p-MLKL, and t-MLKL in lungs mice (n = 3). (D) Representative photomicrographs for lung paraffin sections from mice stained with H&E, PAS, and Masson staining, along with quantification of inflammation scores, PAS-positive cells, and collagen deposition around the airways (n = 5–8). Bars = 50 μm.*p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The methods of asthma model construction, RIPK3 inhibitor GSK872 (Selleck, China) injection, and recombinant TL1A (R&D systems, USA) nasal administration were illustrated in the figures below.

Techniques: Recombinant, Western Blot, Staining

Journal: Respiratory research

Article Title: Necroptosis plays a role in TL1A-induced airway inflammation and barrier damage in asthma.

doi: 10.1186/s12931-024-02900-4

Figure Lengend Snippet: Fig. 6 TL1A-induced necroptosis affects airway epithelial intercellular adhesion. (A) Volcano plot of RNA-seq from OVA-treated lung tissues between MLKL WT (n = 3) and knockout (n = 3) mice. (P < 0.05, log|FC|≥1) (B) Bubble map of KEGG pathway enrichment. (C) HBE cells were transfected with siTL1A and then stimulated by TSZ for 4 h. Occludin and zonulin-1 (ZO-1) expression were determined by Western blot (n = 3). (D) Representative images of MU C5AC, ZO-1, and occludin immunohistochemical staining in lungs of mice (n = 5). (E) Western blot analysis of occludin and ZO-1 in lungs of mice (n = 3). (F) Representative images of MUC5AC, ZO-1, and occludin immunohistochemical staining in lungs of mice (n = 5). (G) Western blot analysis of occludin and ZO-1 in lungs of mice (n = 3). Bars = 50 μm.*p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The methods of asthma model construction, RIPK3 inhibitor GSK872 (Selleck, China) injection, and recombinant TL1A (R&D systems, USA) nasal administration were illustrated in the figures below.

Techniques: RNA Sequencing, Knock-Out, Transfection, Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: Respiratory research

Article Title: Necroptosis plays a role in TL1A-induced airway inflammation and barrier damage in asthma.

doi: 10.1186/s12931-024-02900-4

Figure Lengend Snippet: Fig. 7 TL1A-induced necroptosis impacts cell junction molecules via NF-kB activation. (A) Human protein-protein interaction (PPI) network of necrop tosis, NF-κB, and cell adhesion molecules. (B) Mouse PPI network of necroptosis, NF-κB, and cell adhesion molecules. (C) HBE cells were transfected with siTL1A and then stimulated by TSZ for 4 h. Phosphorylation of p65 and IκBα expression were determined by Western blot. (D, E) Western blot analysis of p-p65, p65, p-IκBα, and IκBα in lungs of mice. (F) HBE cells were treated with the IκBα inhibitor BAY 11-7082 for 1 h before TSZ for 4 h. The expression of p-p65, p-IκBα, ZO-1, and occludin was determined by Western blot. N = 3. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The methods of asthma model construction, RIPK3 inhibitor GSK872 (Selleck, China) injection, and recombinant TL1A (R&D systems, USA) nasal administration were illustrated in the figures below.

Techniques: Activation Assay, Transfection, Phospho-proteomics, Expressing, Western Blot

Journal: Cellular & molecular immunology

Article Title: TL1A and IL-18 synergy promotes GM-CSF-dependent thymic granulopoiesis in mice.

doi: 10.1038/s41423-024-01180-8

Figure Lengend Snippet: Fig. 2 In vivo administration of TL1A + IL-18 results in acute thymic atrophy and increased thymic neutrophil numbers in neonatal and adult mice. A Schematic of the experimental model of TL1A + IL-18 injection in neonatal mice. Wild-type (WT) neonatal mice (P3) were IP injected with either PBS (vehicle) or TL1A [250 ng/day] + IL-18 [100 ng/day] for 4 consecutive days in a final volume of 20 µl. B Schematic of the experimental model of TL1A + IL-18 injection in adult mice (8 weeks old). WT mice were IP injected with either vehicle (PBS) or TL1A [1 µg/ day] + IL-18 [750 ng/day] for 4 consecutive days in a final volume of 200 µl. C Body weights of neonates (P7) injected with either PBS (vehicle) or TL1A + IL-18 (n = 10). Data representative of one of at least five independent experiments are shown. D Pictures of thymuses from neonates (P7) injected with either PBS (vehicle) or TL1A + IL-18. E Thymic cellularity of neonates (P7) injected with either PBS (vehicle) or TL1A + IL-18. (n = 10). Data representative of one of at least five independent experiments are shown. F Quantification of neutrophil numbers in neonatal mice (P7) injected with PBS (gray) or TL1A + IL-18 (blue). Neutrophils were defined as Lin-CD11b+Ly-6G+ cells. The number of neutrophils in the complete thymus was quantified (n = 10). Data are representative of one of at least five independent experiments. G Body weights of adult model mice on Day 5 (D5) that were injected with either PBS (vehicle) or TL1A + IL-18 (n = 10). Data representative of one of at least five independent experiments are shown. H Pictures of thymuses from adults (D5) injected with either PBS (vehicle) or TL1A + IL-18. I Thymic cellularity of adult thymuses (D5) injected with either PBS (vehicle) or TL1A + IL-18 (n = 10). Data representative of one of at least five independent experiments are shown. J Quantification of the neutrophil numbers in adult mice (D5) injected with PBS (gray) or TL1A + IL-18 (blue). Neutrophils were defined as Lin-CD11b+Ly-6G+ cells. Neutrophil numbers were quantified from complete thymuses (n = 8). Data representative of one of at least five independent experiments are shown. Statistics: (C, E, G, I) Unpaired t test with Welch’s correction. E–J The error bars represent the SDs. F–J The Mann–Whitney U test was performed, as the standard deviations were different between treatment groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: TL1A (R&D Systems, #DY1896-05) and IL-18 (Thermo Fisher #88-50618- 88) ELISAs were performed according to the manufacturer’s instructions.

Techniques: In Vivo, Injection, MANN-WHITNEY

Journal: Cellular & molecular immunology

Article Title: TL1A and IL-18 synergy promotes GM-CSF-dependent thymic granulopoiesis in mice.

doi: 10.1038/s41423-024-01180-8

Figure Lengend Snippet: Fig. 3 Thymic neutrophils develop and mature in situ in the neonatal thymic organ culture, and NOTCH restricts their development. A UMAP representation of scRNAseq cell cycle analysis of the three neutrophil clusters. Phase G2/M cells are shown in green. Phase G1 cells are shown in red. Phase S cells are shown in blue. The black arrow highlights neutrophils in phase G2/M (undergoing mitosis), defined as “pre-Neutrophils” (preNeu) in Fig. 1F. B Slingshot trajectory analysis of thymic neutrophils in organ culture. A trajectory from preNeu →imm. Neu →mat. Neu was identified. C Pseudotime analysis (“Slingshot” package) of genes and transcription factors involved in thymic neutrophil development. Kinetics of neutrophil numbers in the NTOC lobes (D) and supernatant (E) during the 6 days of culture with different cytokine treatments. (n = 3). Data representative of one of at least three independent experiments are shown. Error bars represent the SEM. F Heatmap of genes associated with neutrophil maturation. G Flow cytometry characterization of the expression of CD62L, CXCR4, and CD101 in newly generated neutrophils in the NTOC lobes from Day 3 to Day 6 after treatment with TL1A + IL-18. Histograms were set in modal mode. (n = 3). Data representative of one of at least three independent experiments are shown. H EM images from magnetically isolated neonatal bone marrow neutrophils and sorted Ly-6G+ cell NTOC supernatant treated with TL1A + IL-18 on Day 6. The different nuclear morphologies reveal distinct maturation stages of neutrophil development. I NOTCH negatively regulates neutrophil development in the thymus. NTOCs were treated with (1) control (PBS), (2) 1 µM, (3) 5 µM, or (4) 10 µM of the γ-secretase inhibitor LY411575, which prevents NOTCH signaling activation. (n = 3). Data representative of one out of three independent experiments are shown. The error bars represent the SDs. Statistics: (I) One-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. TEM transmission electron microscopy

Article Snippet: TL1A (R&D Systems, #DY1896-05) and IL-18 (Thermo Fisher #88-50618- 88) ELISAs were performed according to the manufacturer’s instructions.

Techniques: In Situ, Organ Culture, Cell Cycle Assay, Flow Cytometry, Expressing, Generated, Isolation, Control, Activation Assay, Transmission Assay, Electron Microscopy

Journal: Cellular & molecular immunology

Article Title: TL1A and IL-18 synergy promotes GM-CSF-dependent thymic granulopoiesis in mice.

doi: 10.1038/s41423-024-01180-8

Figure Lengend Snippet: Fig. 5 Thymic neutrophils can phagocytose, produce ROS, migrate, and form NETs similar to benchmark peritoneal neutrophils. A Schematic of the experimental design used to assess the ROS production capacity, phagocytosis, migration, and NET formation of thymic neutrophils compared to those of peritoneal neutrophils. Thymic neutrophils were isolated from NTOC supernatants treated for 6 days with TL1A + IL-18. Adult peritoneal neutrophils were isolated 4 h after IP injection of 1 ml of 3% Brewer thioglycolate. Neutrophils were purified using an EasySep™Mouse Neutrophil Enrichment Kit. Isolated peritoneal and thymic neutrophils were treated with PBS, eBioscience™Cell Stimulation Cocktail (1:500), DHR 123 [5 µM], pHrodo particles from S. aureus [1 mg/mL], cytochalastin D [2 µM], DMSO, LPS [4 µg/ml] from Klebsiella pneumoniae, and ionomycin [2.5 µg/ml]. B ROS production assay. Both TL1A- and IL-18-induced NTOC-derived and peritoneal neutrophils were treated with DHR 123 [5 µM] at 37 °C and 5% CO2 for 30 min. The fluorescence intensities were measured in channel B530 and are displayed as histograms. The Y-axes are normalized to the mode. Data representative of one of two experiments are shown. C Heatmap of manually curated neutrophil ROS-related genes. D Phagocytosis assay. TL1A + IL-18-induced, NTOC-derived, and peritoneal neutrophils were incubated with PE-conjugated pHrodo particles from S. aureus for 60 min and treated with cytochalastin D [2 µM] to inhibit phagocytosis. The number of engulfment events was normalized to the cell count. Data representative of one of two experiments are shown. E Heatmap of manually curated neutrophil phagocytosis genes. F Migration assay. TL1A + IL-18-induced, NTOC-derived, and peritoneal neutrophils were seeded at 150,000 cells/well on top of a 12-well Transwell plate with 5.0 µm pores and incubated with the neutrophil chemoattractants CXCL1 [50 ng/ mL] and fMLP [10 µM] at 37 °C and 5% CO2 for 90 min. The migrated cells were counted in a BD FACSVerse™Cell Analyzer. Data representative of one of two experiments are shown. G Heatmap of manually curated neutrophil migration-related genes. H Confocal images of thymic neutrophils isolated from the supernatants of TL1A + IL-18-stimulated NCTs (Day 6) and treated with DMSO (vehicle) or ionomycin for 4 h. The cells were stained with DAPI, SYTOX Green, and antibodies for detecting neutrophil elastase and citrullinated histone 3 (citH3). The scale bar represents 10 µM. (n = 3). Data representative of one of three independent experiments are shown. I Heatmap representation of the genes associated with primary/azurophilic, secondary/specific, tertiary/gelatinase or secretory granule release by NTOC-derived neutrophils. Statistics: (F) One-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. C, E, G The Y-axes were subjected to hierarchical clustering. NET Neutrophil extracellular trap

Article Snippet: TL1A (R&D Systems, #DY1896-05) and IL-18 (Thermo Fisher #88-50618- 88) ELISAs were performed according to the manufacturer’s instructions.

Techniques: Migration, Isolation, Injection, Cell Stimulation, Derivative Assay, Phagocytosis Assay, Incubation, Cell Counting, Staining