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

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A is an epithelial cytokine expressed in alveolar epithelium and airway basal cells in human healthy and asthmatic lungs. (A) Single-cell RNA-seq analysis of TNFSF15 ( TL1A ) expression in the LungMAP single-cell human lung atlas. Uniform manifold projection (UMAP) plots show the clustering of 347,970 lung cells (10 single-cell datasets, 148 normal human lung samples from 104 donors: adult, child, and adolescent). Results are visualized using ShinyCell and are based upon data generated by the LungMAP Consortium and downloaded from http://www.lungmap.net . (B and C) Single-cell RNA-seq analysis of TNFSF15 ( TL1A ) expression in epithelial cells from human healthy (B) and asthmatic (C) lungs. t-SNE plots show clustering of 26,154 epithelial cells in upper and lower airways and lung parenchyma in healthy lungs (B; 17 human samples: 6 alveoli and parenchyma, 9 bronchi, 2 nasal), and 25,146 epithelial cells from lower airways in healthy and asthmatic lungs (C; 12 human samples: 15,033 cells from 6 asthma bronchi; 10,113 cells from 6 control bronchi). t-SNE plots were extracted from data obtained by the human lung single-cell atlas and downloaded from https://asthma.cellgeni.sanger.ac.uk .

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: RNA Sequencing, Expressing, Generated, Control

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Single-cell RNA-seq analysis of IL33 and TSLP expression in human lungs and gating strategy for analysis of mouse lung epithelial cells by flow cytometry. (A and B) Single-cell RNA-seq analysis of IL33 and TSLP expression in epithelial cells from human healthy (A) and asthmatic (B) lungs. t-SNE plots show clustering of 26,154 epithelial cells in upper and lower airways and lung parenchyma in healthy lungs (A; 17 human samples: 6 alveoli and parenchyma, 9 bronchi, 2 nasal), and 25,146 epithelial cells from lower airways in healthy and asthmatic lungs (B; 12 human samples: 15,033 cells from 6 asthma bronchi; 10,113 cells from 6 control bronchi). t-SNE plots were extracted from data obtained by the human lung single-cell atlas , and downloaded from https://asthma.cellgeni.sanger.ac.uk . (C) Gating strategy of Epcam + epithelial cells and CD31 + endothelial cells in the lung of a naïve WT mouse. (D and E) Immunohistofluorescence staining of lung tissue sections (naïve wild type C57BL/6J mouse, steady state) with two distinct rat IgG1 isotype controls (rat IgG1 clone eBRG1, D, red; rat IgG1 clone RB40.34, E, red) for the anti-TL1A antibody (rat IgG1, MAB7441, clone 293327). Double staining was performed with antibodies against RAGE (D, green) or IL-33 (E, green). Images are representative of two independent experiments. Scale bar, 10 μm.

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Control, Immunohistofluorescence, Staining, Double Staining

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A is expressed in mouse alveolar epithelium at steady state. (A) Visualization of Tnfsf15 (TL1A) expressing cells in the LungMAP single-cell mouse lung atlas. UMAP plots show the clustering of 95,658 lung cells (17 samples from late developmental stage to postnatal day 28). The different cell types in the lungs of naïve mice are indicated on the left. Results are visualized using ShinyCell and are based upon data generated by the LungMAP Consortium and downloaded from http://www.lungmap.net . (B) Single-cell RNA-seq analysis of Tnfsf15/TL1A and Il33 gene expression in mouse lung epithelium. UMAP plots show clustering and cell type annotation of 12,536 mouse lung epithelial cells (seven samples from the emergence of the alveolus to postnatal day 28) . The number and percentage of epithelial cells expressing Tnfsf15/TL1A , Il33 , or both are indicated on the right. Results are visualized using ShinyCell and are based upon data obtained by and downloaded from http://www.lungmap.net . (C) Flow cytometry analysis of cell surface TL1A expression on live CD31 + CD45 − endothelial cells and Epcam + CD31 − CD45 − epithelial cells in the lung of a naïve wild type C57BL/6J mouse at steady state. (D and E) Immunohistofluorescence staining of lung tissue sections (naïve wild type C57BL/6J mouse, steady state) with antibodies against TL1A (D and E) and RAGE (D) or IL-33 (E) proteins. A tyramide signal amplification (TSA)-based immunofluorescence method was used to detect TL1A-expressing cells in situ. Images are representative of two independent experiments. Scale bar, 10 μm.

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: Expressing, Generated, RNA Sequencing, Gene Expression, Flow Cytometry, Immunohistofluorescence, Staining, Amplification, Immunofluorescence, In Situ

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: High throughput proteomic analyses of lung ILC2s stimulated ex vivo with IL-33 and/or TL1A. (A) Flow cytometry of cultured lung ILC2s ex vivo. Representative histograms of ST2, CD90.2, Sca-1, CD25, ICOS, KLRG1, and DR3 expression at the surface of cultured ILC2s, 3 days after ILC2 cell isolation from the lung and ex vivo culture in the presence of IL-2. Phenotypic analysis was performed on live Lin – CD45 + cells. (B–D) Large-scale label-free proteomic analyses of mouse lung ILC2s after ex vivo overnight stimulation with rIL-2 ± rIL-33 ± rTL1A. Volcano plots of IL-33-stimulated ILC2s (B) or TL1A-stimulated ILC2s (C) compared with non-stimulated cells (NS; in culture with IL-2 alone). Volcano plot of IL-33/TL1A-stimulated ILC2s compared to IL-33-stimulated cells (D). Statistical analysis of protein abundance values was performed from different biological replicate experiments ( n = 6 for NS and IL33 stimulation; n = 3 for TL1A and IL33/TL1A stimulations), using a Student’s t test (log 10 P value, vertical axis). Proteins found as significantly over or under-expressed (P < 0.05 and abs[log 2 fold change] >1) are shown in black. Representative examples of proteins found modulated in each comparison are shown in color. (E) Flow cytometry of cultured lung ILC2s after 14 h of co-stimulation with IL-33 and TL1A in the presence of IL-2 (ILC2 culture used in ). Intracellular cytokine staining revealed that >99% of ILC2s co-expressed IL-9 and IL-13 intracellularly. Phenotypic analysis was performed on live Lin − CD45 + CD90.2 + cells.

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: High Throughput Screening Assay, Ex Vivo, Flow Cytometry, Cell Culture, Expressing, Cell Isolation, Quantitative Proteomics, Comparison, Staining

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A synergizes with IL-33 to induce an IL-9-producing ILC9 phenotype in lung ILC2s. (A and B) Large-scale label-free proteomic analyses of ILC2s isolated from pooled lungs of IL-33-treated Rag2 −/− C57BL/6 J mice and cultured with IL-2 prior to overnight stimulation with rIL-2 ± rIL-33 ± rTL1A. Volcano plot of IL-33/TL1A-stimulated ILC2s (ILC9 cells) compared with nonstimulated cells (NS; in culture with IL-2 alone) (A). Statistical analysis of protein abundance values was performed from different biological replicate experiments ( n = 6 for NS; n = 3 for IL33/TL1A stimulation) using a Student’s t test (log 10 P value, vertical axis). Proteins found as significantly over or under-expressed (P < 0.05 and abs[log 2 fold change] >1) are shown in black. Examples of proteins modulated in both IL-33/TL1A-stimulated ILC2s and IL-33-stimulated ILC2s are shown in blue. Proteins shown in red are representative of molecules specifically modulated in IL-33/TL1A-stimulated ILC2s (A). Heat-map of fold changes of selected proteins in three independent biological replicates (B). (C–K) Analysis of ILC2s isolated from pooled lungs of IL-33-treated Rag2 −/− C57BL/6 J mice , and cultured with IL-2 prior to 14 h stimulation with rIL-2 ± rIL-33 ± rTL1A. Flow cytometry analysis of live Lin − CD45 + cells (C, E, and J), frequency of IL-9 high ILC2s (percentage of live Lin − CD45 + CD90.2 + cells) (D and K), and MFI fold change of IL-9 in ILC2s (E), after cytokines treatment and restimulation by PMA, ionomycin, and brefeldin A (4 h, C–E) or brefeldin A (4 h, J and K). Concentration of IL-9 secreted by ILC2s, measured by ELISA (F). Relative STAT5 mRNA expression levels measured by real-time qPCR (G). Samples were normalized to the expression of HPRT and are shown relative to IL-2-stimulated ILC2s. Immunoblot analysis of activated phosphorylated STAT5 (pSTAT5) and α-tubulin (H) or β-actin (I); Arrowheads indicate the migration of the protein of interest; cropped images. Cultured ILC2s were treated with rIL-2 + rIL-33 + rTL1A and increasing doses of a STAT5 inhibitor (STA5i, CAS 285986-31-4) or control vehicle (DMSO) (I–K). Numbers inside outlined areas (C) indicate percent of cells in the relevant gate. Each symbol represents an individual biological replicate (D–G and K). Data are pooled from six (D and E), six to eight (F) or three (G and K) independent experiments, or are representative of six (C and E) or three (H–J) independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (D–G and K): ns not significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are available for this figure: .

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: Isolation, Cell Culture, Quantitative Proteomics, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Migration, Control

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A synergistically induce IL-9-producing ILC2s ex vivo. (A) Analysis of cultured lung ILC2s 14 h after ex vivo stimulation by rIL-2 (20 ng/ml) ± rIL-33 (20 ng/ml) ± rTL1A (50 ng/ml). Flow cytometry analysis of live Lin − CD45 + cells and frequency of IL-9 high ILC2s (percentage of live Lin − CD45 + CD90.2 + cells) after cytokine treatment and incubation with brefeldin A (4 h), without restimulation by PMA and ionomycin. Numbers inside outlined area indicate percent of cells in the relevant gate and data are representative of eight independent experiments. (B) Concentration of IL-9 secreted by ILC2s treated with rIL-2 (20 ng/ml) and various concentrations of rIL-33 and rTL1A measured by ELISA. (C and D) MFI of nuclear factor IRF4 (C) and flow cytometry (D) of ILC2s 14 h after ex vivo stimulation of cultured ILC2s by rIL-2 (20 ng/ml) ± rIL-33 (20 ng/ml) ± rTL1A (50 ng/ml). Numbers inside outlined areas (D) indicate percent of cells in the relevant gate and data are representative of three independent experiments. (E) Immunoblot analysis of JunB and α-tubulin14 h after cytokine stimulation of lung ILC2s; Arrowheads indicate the migration of the protein of interest; cropped image. Data are representative of three independent experiments. (F–H) Relative mRNA expression levels by real time qPCR, 14 h after cytokine stimulation of lung ILC2s. Samples were normalized to the expression of HPRT and data are expressed relative to IL-2-stimulated ILC2s (F) or relative to HPRT mRNA quantity (G and H). (I and J) Analysis of mouse lung ILC2s 14 h after ex vivo stimulation by rIL-33 + rTL1A ± rIL-2 ± rIL-7 ± rTSLP. Frequency of IL-9 high ILC2s (Lin − CD45 + CD90.2 + cells), after cytokines treatment and re-stimulation by PMA, ionomycin and brefeldin A (4 h, I). Concentration of IL-9 secreted by ILC2s, measured by ELISA (J). (K) Concentration of IL-9 (ELISA) secreted by ILC2s 14 h after ex vivo stimulation by rIL-2 ± rIL-33 ± rIL-4 ± rTGF-β. Each symbol represents an individual biological replicates with n = 2–5 independent experiments (A–C and F–K). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (A, C, and F–J) or Dunnett’s (B and K) multiple-comparisons tests: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. In H, all significant P values are annotated with stars, all other comparisons are not significant. Source data are available for this figure: .

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: Ex Vivo, Cell Culture, Flow Cytometry, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Migration, Expressing

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A induce phenotypic changes in cultured lung ILC2s at the protein and mRNA levels. (A–J) Analysis of mouse lung ILC2s 14 h after ex vivo stimulation by rIL-2 ± rIL-33 ± rTL1A. MFI of the indicated cell surface markers determined by flow cytometry (A, B, D, and E). Relative mRNA expression levels of various genes (C and F–I), including genes characteristic of ILC1s or ILC3s (I), determined by real-time qPCR, 14 h after cytokine stimulation of lung ILC2s. Samples were normalized to the expression of HPRT and data are expressed as relative to HPRT mRNA quantity. Concentration of IL-5 or IL-13 in cell supernatants, measured by ELISA assay (J). Each symbol represents an individual biological replicate from independent experiments (A–J). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t test (B, E, and J) or one-way ANOVA followed by Tukey’s multiple-comparisons test (A, C, D, and F–I): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. In I, all significant P values are annotated with stars, all other comparisons are not significant.

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: Cell Culture, Ex Vivo, Flow Cytometry, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A cooperates with IL-33 for induction of IL-9 high ILC2s in vivo. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B) Gating strategy of IL-9 high IL-5 + IL-13 + ILC2s. (C–I) Flow cytometry of IL-5 + IL-13 + ILC2s gated on live ILCs (Lin − CD45 + CD90.2 + cells) (C) and IL-9 high ILC2s gated on live IL-5 + IL-13 + ILC2s (E), frequency of lung IL-5 + IL-13 + ILC2s among live ILCs (D), IL-9 high ILC2s among live IL-5 + IL-13 + ILC2s (F), and IL-9 high IL-13 + ILC2s among live ILCs (G) or IL-9 high ILCs (H), and concentration of IL-9 in BAL fluids (ELISA assay, I) of WT mice 14 h after a single i.n. administration of PBS or rIL-33 (1 μg) and/or rTL1A (5 μg). Numbers inside outlined areas indicate the percent of cells in the relevant gate and data are representative of two independent experiments (C and E). Each symbol represents an individual mouse and data are pooled from two independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (D) or Dunnett’s (F, G, and I) multiple-comparisons tests: ns, not significant, ** P < 0.01, **** P < 0.0001. (J) Frequency of lung eosinophils (Gr1 low Siglec-F + CD11c − cells) among live CD45 + cells, at day 7 after a single i.n. exposure to rIL-33 or rIL-33 plus rTL1A. Each symbol represents an individual mouse and data are pooled from two independent experiments. Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t test: * P < 0.05. (K and L) Multiphoton imaging (K) and intravital microscopy (L) of whole lungs of INFER IL-9 fluorescent reporter mice, with detection of IL-9-eGFP + ILC2s (green) and staining of blood vessels (red) and collagen fibers (blue), 16–18 h after a single i.n. administration of IL-33/TL1A combination (1 μg rIL-33 plus 5 μg rTL1A). To increase the numbers of lung IL-9 high ILC2s accessible to in vivo imaging, the single i.n. exposure to IL-33/TL1A combination was performed after prior expansion of lung ILC2s by repeated i.p. injections of IL-33 (K and L). Multiphoton image (K) is a 3D reconstitution of stitched images (7 × 7 tiles and 181 z-stack). Time-lapse images (L) illustrate the migratory behavior of IL-9-eGFP + ILC2s. Time in h/min/s. Scale bars: K, 300 μm; L, 20 μm.

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: In Vivo, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Imaging, Intravital Microscopy, Staining, In Vivo Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A synergistically induce IL-9-producing ILC2s in vivo. (A) Gating strategy and representative flow cytometry plots of live lung ILCs (live Lin − CD45 + CD90.2 + cells), live lung IL-5 + IL-13 + ILC2s (live IL-5 + IL-13 + ILCs) and live lung IL-9 high ILC2s (live IL-9 high IL-5 + IL-13 + ILC2s) in vivo in wild type (WT) C57BL/6J mouse, 14 h after a single i.n. administration of rIL-33 (1 μg) and rTL1A (5 μg). (B) Verification of the absence of contamination of the IL-5 + IL-13 + ILC2s and IL-9 high ILC2s populations by TCR + cells (T cells and NKT cells) using anti-TCRβ and anti-TCRγδ antibodies. (C) Confirmation of the expression of IL-5 and IL-13 in live Lin − CD3/TCR − NK1.1 − CD45 + CD90.2 + lung ILCs using antibodies against CD3/TCR and NK1.1 with a different fluorescence from the Lin cocktail (CD4, CD19, CD45R, CD11b, CD11c, Ter119, Ly6G, FcεRI). (D and E) Frequency of lung IL-9 high Lin − cells among live CD45 + cells (D), and flow cytometry of IL-9 high IL-13 + ILC2s (live IL-9 high IL-13 + Lin − CD45 + CD90.2 + cells) (E) of WT mice 14 h after a single i.n. administration of PBS or rIL-33 (1 μg) and/or rTL1A (5 μg). Numbers inside outlined areas indicate the percent of cells in the relevant gate. (F) Frequency of lung IL-9 high Lin − cells among live CD45 + cells of WT mice pretreated with six daily i.p. injections of rIL-33 (days 1–6) prior to one i.n. injection of PBS or rIL-33 and/or rTL1A (day 7). Flow cytometry analyses were performed on day 8. (G) Frequency of IL-9 high ILC2s among live ILCs (Lin − CD45 + CD90.2 + cells) in the lungs of WT mice 6 h after a single i.n. administration of A. alternata extract (12.5 μg), with (αIL-2 mAb) or without (Iso, isotype control mAb) IL-2 blockade. (H and I) Analysis of IL-9 and TL1A release in BAL fluids by ELISA at different time points after the third exposure to A. alternata in a chronic exposure model (repeated i.n. administration of 12.5 μg A. alternata at days 0, 3, and 6). Each symbol represents an individual mouse and data are pooled from two (D and G) or three (F, H, and I) independent experiments. Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (G) or one-way ANOVA followed by Dunnett’s multiple-comparison test (D, F, H, and I): * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: In Vivo, Flow Cytometry, Expressing, Fluorescence, Injection, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Endogenous IL-9-producing ILC2s accumulate around blood vessels after IL33/TL1A treatment in vivo. IL9-eGFP + ILC2s (green), blood vessels (Evans Blue/red), and collagen fibers (second harmonic generation/blue) were visualized by multiphoton imaging in the cleared lung of INFER IL9 fluorescent reporter mice 16–18 h after administration of IL33/TL1A combination. 360° rotation of a 3D static representation at a frame rate of 25 fps (500 frames per 20 sec).

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: In Vivo, Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Endogenous IL-9-producing ILC2s migrate along collagen fibers after IL33/TL1A treatment in vivo. IL9-eGFP + ILC2s (green), blood vessels (Evans Blue/red), and collagen fibers (second harmonic generation/blue) were visualized by lung intravital multiphoton imaging of INFER IL9 fluorescent reporter mice 16–18 h after administration of IL33/TL1A combination. Time in h/min/s. Playback speed: 600.

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: In Vivo, Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A functions as an epithelial alarmin rapidly released after allergen exposure. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B–F) Analysis of TL1A and IL-33 release in BAL fluids after a single allergen exposure. TL1A (B and E), IL-33 (C and F), and LDH (D) levels in BAL fluids were determined by ELISA (B, C, E, and F) or LDH (D) assays, 15 min (B–D) or at different time points (E and F) after a single i.n. administration of A. alternata extract (12.5 μg). Each symbol represents an individual mouse and data are pooled from two independent experiments (B–F). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (B–D) or Dunnett’s (E and F) multiple-comparisons tests: ** P < 0.01, *** P < 0.001, **** P < 0.0001. (G–K) Analysis of TL1A release in cell supernatants after exposure of TL1A-expressing cells to A. alternata or bee venom phospholipase A2 (PLA2). U2OS epithelial cells transfected with a mouse TL1A-Flag expression vector (mTL1A-Flag vector) or control vector were analyzed by indirect immunofluorescence microscopy with anti-mTL1A and anti-Flag antibodies (G). Scale bar, 20 μm. TL1A (H and J) and LDH (I and K) levels in cell supernatants were determined by ELISA (H and J) or LDH cytotoxicity assays (I and K) 15 min after treatment with A. alternata extract ( A. alternata , H and I) or 1 h after treatment with bee venom PLA2 (J and K). NT, not treated. Each symbol represents an individual biological replicate and data are pooled from three independent experiments (H–K). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (treatment versus NT): ** P < 0.01, **** P < 0.0001.

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation, Control, Immunofluorescence, Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A is important for early induction of IL-9 high ILC2s after allergen exposure. (A) Treatment schedule of naïve WT mice. (B) IL-9 mRNA levels in the lungs analyzed by qPCR at different time points after a single allergen exposure. Data are expressed as relative to IL-9 mRNA levels in mice treated with PBS. (C–H) Flow cytometry and frequency of IL-9 high Lin − cells among live CD45 + cells (C and D) and IL-9 high ILC2s among live ILCs (Lin − CD45 + CD90.2 + cells) (E and F), flow cytometry (G), and MFI of IRF4 expression in ILC2s (H), in the lungs of WT mice 6 h after a single i.n. administration of A. alternata extract (12.5 μg), with (αTL1A mAb) or without (Iso, isotype control mAb) TL1A blockade. Numbers inside outlined areas indicate the percent of cells in the relevant gate (C, E, and G) and data are representative of two (G) or three (C and E) independent experiments. Each symbol represents an individual mouse and data are pooled from three (D and F) or two (B and H) independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (B) or unpaired two-tailed Student’s t tests (D, F, and H): ns, not significant, *** P < 0.001, **** P < 0.0001.

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: Flow Cytometry, Expressing, Control, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: ILC9 cells have an increased capacity to initiate IL-5-dependent allergic airway inflammation. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice by a single i.v. adoptive cell transfer of classical IL-33-activated ILC2s (ILC2) or IL-33/TL1A-activated ILC2s (ILC9). (B–H) Flow cytometry (B and D) and frequency of eosinophils (Gr1 low Siglec-F + CD11c − cells) among live CD45 + cells from BALF (C and F) or lung (E and G), and number of Red5 + ILC2s or ILC9s in total lung of mice (H), at day 7 after a single i.v. adoptive transfer of 5 × 10 5 ILC2s or ILC9s in separate host mice. Adoptively transferred ILC2s and ILC9s were prepared from Rag2 −/− mice ( Il5 +/+ cells) (B–E) or Red5 mice ( Il5 −/− cells) (F–H). Control mice received an intravenous injection of PBS. Red5 + cells indicate the activity of the Il5 promoter. Each symbol represents an individual mouse and data are representative (B and D) or pooled (C and E–H) from two independent experiments. (I–K) Live imaging of ILC2s and ILC9 cells in the lung. Lung intravital microscopy was performed 1–4 h after adoptive transfer of 6 × 10 5 of each cell type in the same host (green, classical IL-33-activated ILC2s-CFSE + ; red, IL-33/TL1A-activated ILC9 cells-CTO + ) (I). Imaging of the migratory behavior of ILC2s and ILC9 cells in the lung (J) and cell quantification from lung intravital microscopy data (K). Time-lapse images, 2 h after adoptive cell transfer (J). A maximum intensity projection of stitched images (2 × 2 tiles and 18 z-stack) is shown (K). Time in h/min/s. Scale bars: J, 20 μm; K, 100 μm. Lung intravital microscopy data are representative (J and K) or analyzed (K) from three adoptive transfer experiments on four mice. Data are expressed as mean (±SEM) with P values determined by paired two-tailed Student’s t test (K) or one-way ANOVA followed by Tukey’s multiple-comparisons test (C and E–H): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: Flow Cytometry, Adoptive Transfer Assay, Control, Injection, Activity Assay, Imaging, Intravital Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Adoptively transferred ILC2s and ILC9s are equally recruited to the lung and exhibit an ameboid-like mode of migration. IL-33-activated ILC2s (CFSE/green), IL33/TL1A-activated ILC9s (CTO/red), blood vessels (Evans Blue/dark blue), and collagen fibers (second harmonic generation/light blue) were observed by lung intravital multiphoton imaging 2 h after intravenous adoptive transfer (6 × 10 5 cells). Time in h/min/s. Playback speed: 600.

Article Snippet: TL1A levels in BAL fluids and culture supernatants were determined with a modified mouse duoset TL1A ELISA (R&D Systems) by using the function-blocking anti-TL1A mAb L4G6 (clone L4G6, #EMI006; Kerafast) as a capture antibody and the neutralizing anti-TL1A mAb 820446 (clone 820446, # MAB1896; R&D Systems) for detection.

Techniques: Migration, Imaging, Adoptive Transfer Assay

Journal: Frontiers in Immunology

Article Title: Targeting TL1A/DR3 Signaling Offers a Therapeutic Advantage to Neutralizing IL13/IL4Rα in Muco-Secretory Fibrotic Disorders

doi: 10.3389/fimmu.2021.692127

Figure Lengend Snippet: TL1A induces mucus production when administered in isolation into the airways. (A) Schematic representation of the protocol used. Briefly, C57BL/6 mice were induced with 10μg of recombinant TL1A i.t. on two successive days. Mice were euthanized 24h after the last i.t. injection, and lungs were harvested for RNAseq analysis, PAS, ClcA1 and MUC5AC stains. (B) Heatmap of the top differentially regulated genes involved in mucus production, upregulated in the lungs of TL1A-induced mice. (C) Top panel: PAS stain of mucus produced in the lungs and quantified using Image Pro Premier and graphed as %PAS+ bronchial epithelial cells per lobe (right panel). Mid and Lower panels: Immunofluorescence stains of CLcA1 (green), MUC5AC (red) and nuclear DAPI (blue). All results representative of three experiments with four to six mice per group. **p < 0.005.

Article Snippet: Activity of recombinant protein: WT C57BL/6 mice, RAG2-deficient mice, and RAG2γc-deficient mice, were given 10 μg of recombinant mouse TL1A (R&D Systems, Minneapolis, MN) or PBS intratracheally on days 1 and 2 and sacrificed for analyses one day later on day 3.

Techniques: Isolation, Recombinant, Injection, Staining, Produced, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Targeting TL1A/DR3 Signaling Offers a Therapeutic Advantage to Neutralizing IL13/IL4Rα in Muco-Secretory Fibrotic Disorders

doi: 10.3389/fimmu.2021.692127

Figure Lengend Snippet: Interrupting TL1A/DR3 signaling decreases mucus production in allergen-induced asthma. (A) Schematic representation of protocol used. Briefly, WT littermates and DR3-deficient mice on the C57BL/6 x 129 background were sensitized i.n. on day 0, 7, and 14 with 200 and 100 μg house dust mite extract protein in PBS, followed by chronic i.n. challenges of 50 μg of HDM protein administered twice a week for the following four weeks. Analyses were performed 24 hours after the last challenge. For neutralization of TL1A-DR3 interactions, mouse DR3-Fc or isotype control IgG were administered i.p. to WT C57BL/6 mice after the initial sensitization period starting at day 14 and were given every three days until the end of the experiment (100 μg/injection/mouse). (B) PAS stain of mucus produced in the lungs and quantified using Image Pro Premier. (C) Inflammation assessed by H&E stain (top panels), collagen deposition assessed by trichrome stain (middle panels), and smooth muscle hypertrophy assessed by αSMA immunofluorescence stain (red) (bottom panels) on lung biopsies of WT C57BL/6 mice after treatment with either IgG or DR3-Fc. Quantifications for each parameter, completed using Image Pro Premier, are shown to the right, including untreated WT C57BL/6 and DR3 -/- mice (images not shown). All results representative of three experiments with five mice per group. *p < 0.05, ** < 0.005, ***p < 0.0005, ****p < 0.00005.

Article Snippet: Activity of recombinant protein: WT C57BL/6 mice, RAG2-deficient mice, and RAG2γc-deficient mice, were given 10 μg of recombinant mouse TL1A (R&D Systems, Minneapolis, MN) or PBS intratracheally on days 1 and 2 and sacrificed for analyses one day later on day 3.

Techniques: Neutralization, Control, Injection, Staining, Produced, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Targeting TL1A/DR3 Signaling Offers a Therapeutic Advantage to Neutralizing IL13/IL4Rα in Muco-Secretory Fibrotic Disorders

doi: 10.3389/fimmu.2021.692127

Figure Lengend Snippet: TL1A muco-secretory activity is dependent on IL13/IL4Rα signaling. (A) Schematic representation of the protocol used, as previously described in Figure 1 . (B) Top panel: PAS stain of mucus produced in the lungs and quantified using Image Pro Premier (right panel). Mid and Lower panels: Immunofluorescence stains of CLcA1 (green), MUC5AC (red) and nuclear DAPI (blue). (C) Inflammation assessed by H&E stain (top panels), collagen deposition assessed by trichrome stain (middle panels), and smooth muscle hypertrophy assessed by αSMA immunofluorescence stain (red) (bottom panels) on lung biopsies of BALB/c and IL4Rα-deficient mice induced with PBS or TL1A. Quantification for each parameter is done using Image Pro Premier. All results representative of three experiments with four to six mice per group. ns, not significant, *p < 0.05, ** < 0.005.

Article Snippet: Activity of recombinant protein: WT C57BL/6 mice, RAG2-deficient mice, and RAG2γc-deficient mice, were given 10 μg of recombinant mouse TL1A (R&D Systems, Minneapolis, MN) or PBS intratracheally on days 1 and 2 and sacrificed for analyses one day later on day 3.

Techniques: Activity Assay, Staining, Produced, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Targeting TL1A/DR3 Signaling Offers a Therapeutic Advantage to Neutralizing IL13/IL4Rα in Muco-Secretory Fibrotic Disorders

doi: 10.3389/fimmu.2021.692127

Figure Lengend Snippet: TL1A induces mucus production independently of adaptive immunity, bystander to IL13 production by ILC2. (A) Schematic representation of the protocol used, as previously described in Figure 1 . (B) Top panel: PAS stain of mucus produced in the lungs of RAG -/- and RAGγc -/- mice induced with PBS or TL1A and quantified using Image Pro Premier (right graph). Lower panels: Immunofluorescence stains of MUC5AC (red) and nuclear DAPI (blue). (C) DR3 staining on ILC2 assessed by flow cytometry (DR3 in blue and Isotype in red). (D) Number of lung ILC2 in the left lobe of RAG -/- mice induced with TL1A. (E) IL13 levels assessed by ELISA, in the supernatants of ILC2 cultured in the presence of IL2, IL7, and IL33 and stimulated with or without TL1A. All results representative of three experiments with four to six mice per group, or three individual replicate cultures. *p < 0.05, **p < 0.005.

Article Snippet: Activity of recombinant protein: WT C57BL/6 mice, RAG2-deficient mice, and RAG2γc-deficient mice, were given 10 μg of recombinant mouse TL1A (R&D Systems, Minneapolis, MN) or PBS intratracheally on days 1 and 2 and sacrificed for analyses one day later on day 3.

Techniques: Staining, Produced, Immunofluorescence, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Frontiers in Immunology

Article Title: Targeting TL1A/DR3 Signaling Offers a Therapeutic Advantage to Neutralizing IL13/IL4Rα in Muco-Secretory Fibrotic Disorders

doi: 10.3389/fimmu.2021.692127

Figure Lengend Snippet: Graphical Abstract. Upon epithelial injury, such as exposure to allergens, TL1A is produced and acts on ILC2 via DR3 to promote IL13 secretion, leading to mucus production. Created with BioRender.com .

Article Snippet: Activity of recombinant protein: WT C57BL/6 mice, RAG2-deficient mice, and RAG2γc-deficient mice, were given 10 μg of recombinant mouse TL1A (R&D Systems, Minneapolis, MN) or PBS intratracheally on days 1 and 2 and sacrificed for analyses one day later on day 3.

Techniques: Produced

Journal: PLoS ONE

Article Title: The Somatostatin Analogue Octreotide Inhibits Growth of Small Intestine Neuroendocrine Tumour Cells

doi: 10.1371/journal.pone.0048411

Figure Lengend Snippet: Gene ontology of 25 selected genes from microarray analyses.

Article Snippet: Monoclonal mouse anti-human ANXA1 (1∶5000, BD-Transduction Laboratories, Franklin Lakes, NJ, USA), polyclonal rabbit anti-human ARHGAP18 (1∶250, Abgent, San Diego, CA, USA), polyclonal mouse anti-human EMP1 (1∶250, Abnova, Taipei, Taiwan), polyclonal rabbit anti-human GDF15 (1∶250, Atlas Antibodies, Stockholm, Sweden), polyclonal goat anti-human TGFBR2 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal goat anti-human TNFSF15 (1∶500, R&D Systems, Minneapolis, MN, USA), polyclonal rabbit anti-human SSTR1 (1∶5000, Santa Cruz Biotechnology), polyclonal rabbit anti-human SSTR2 (1∶4000, Thermo Scientific), polyclonal rabbit anti-human SSTR3 and SSTR5 (1∶4000 and 1∶5000, gifts from Frank Leu , polyclonal goat anti-human β-actin and HRP donkey anti-goat (1∶5000, Santa Cruz Biotechnology) and HRP anti-mouse and anti-rabbit (1∶5000, Amersham Biosciences, Buckinghamshire, England) antibodies were used to detect the different proteins.

Techniques: Microarray, Protein Binding, Activity Assay, Transduction, Binding Assay, Membrane, Migration, De-Phosphorylation Assay, Phospho-proteomics

Journal: PLoS ONE

Article Title: The Somatostatin Analogue Octreotide Inhibits Growth of Small Intestine Neuroendocrine Tumour Cells

doi: 10.1371/journal.pone.0048411

Figure Lengend Snippet: ANXA1, ARHGAP18, EMP1, GDF15, TGFBR2 and TNFSF15 were analysed using total RNA at 1 week (wk), 4 months, 10 months and 16 months (mo) of culture by QRT-PCR. Results were plotted using the 2 −ΔΔCt method with β-actin expression (set to 1) from each individual sample as endogenous reference. Plotted results are means ± SD for triplicate wells. Significance was calculated by Two-Way ANOVA followed by Bonferroni test; comparing with untreated CNDT2.5 cells. *** = p <0.001.

Article Snippet: Monoclonal mouse anti-human ANXA1 (1∶5000, BD-Transduction Laboratories, Franklin Lakes, NJ, USA), polyclonal rabbit anti-human ARHGAP18 (1∶250, Abgent, San Diego, CA, USA), polyclonal mouse anti-human EMP1 (1∶250, Abnova, Taipei, Taiwan), polyclonal rabbit anti-human GDF15 (1∶250, Atlas Antibodies, Stockholm, Sweden), polyclonal goat anti-human TGFBR2 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal goat anti-human TNFSF15 (1∶500, R&D Systems, Minneapolis, MN, USA), polyclonal rabbit anti-human SSTR1 (1∶5000, Santa Cruz Biotechnology), polyclonal rabbit anti-human SSTR2 (1∶4000, Thermo Scientific), polyclonal rabbit anti-human SSTR3 and SSTR5 (1∶4000 and 1∶5000, gifts from Frank Leu , polyclonal goat anti-human β-actin and HRP donkey anti-goat (1∶5000, Santa Cruz Biotechnology) and HRP anti-mouse and anti-rabbit (1∶5000, Amersham Biosciences, Buckinghamshire, England) antibodies were used to detect the different proteins.

Techniques: Quantitative RT-PCR, Expressing

Journal: PLoS ONE

Article Title: The Somatostatin Analogue Octreotide Inhibits Growth of Small Intestine Neuroendocrine Tumour Cells

doi: 10.1371/journal.pone.0048411

Figure Lengend Snippet: Result of Immunohistochemistry on paraffin embedded SI-NET specimens.

Article Snippet: Monoclonal mouse anti-human ANXA1 (1∶5000, BD-Transduction Laboratories, Franklin Lakes, NJ, USA), polyclonal rabbit anti-human ARHGAP18 (1∶250, Abgent, San Diego, CA, USA), polyclonal mouse anti-human EMP1 (1∶250, Abnova, Taipei, Taiwan), polyclonal rabbit anti-human GDF15 (1∶250, Atlas Antibodies, Stockholm, Sweden), polyclonal goat anti-human TGFBR2 (1∶250, Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal goat anti-human TNFSF15 (1∶500, R&D Systems, Minneapolis, MN, USA), polyclonal rabbit anti-human SSTR1 (1∶5000, Santa Cruz Biotechnology), polyclonal rabbit anti-human SSTR2 (1∶4000, Thermo Scientific), polyclonal rabbit anti-human SSTR3 and SSTR5 (1∶4000 and 1∶5000, gifts from Frank Leu , polyclonal goat anti-human β-actin and HRP donkey anti-goat (1∶5000, Santa Cruz Biotechnology) and HRP anti-mouse and anti-rabbit (1∶5000, Amersham Biosciences, Buckinghamshire, England) antibodies were used to detect the different proteins.

Techniques: Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A functions as an epithelial alarmin rapidly released after allergen exposure. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B–F) Analysis of TL1A and IL-33 release in BAL fluids after a single allergen exposure. TL1A (B and E), IL-33 (C and F), and LDH (D) levels in BAL fluids were determined by ELISA (B, C, E, and F) or LDH (D) assays, 15 min (B–D) or at different time points (E and F) after a single i.n. administration of A. alternata extract (12.5 μg). Each symbol represents an individual mouse and data are pooled from two independent experiments (B–F). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (B–D) or Dunnett’s (E and F) multiple-comparisons tests: ** P < 0.01, *** P < 0.001, **** P < 0.0001. (G–K) Analysis of TL1A release in cell supernatants after exposure of TL1A-expressing cells to A. alternata or bee venom phospholipase A2 (PLA2). U2OS epithelial cells transfected with a mouse TL1A-Flag expression vector (mTL1A-Flag vector) or control vector were analyzed by indirect immunofluorescence microscopy with anti-mTL1A and anti-Flag antibodies (G). Scale bar, 20 μm. TL1A (H and J) and LDH (I and K) levels in cell supernatants were determined by ELISA (H and J) or LDH cytotoxicity assays (I and K) 15 min after treatment with A. alternata extract ( A. alternata , H and I) or 1 h after treatment with bee venom PLA2 (J and K). NT, not treated. Each symbol represents an individual biological replicate and data are pooled from three independent experiments (H–K). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (treatment versus NT): ** P < 0.01, **** P < 0.0001.

Article Snippet: A pCMV6-mTL1A (Tnfsf15, NM_177371 ) expression vector allowing ectopic expression of the mouse TL1A protein (252 aa) tagged at its C-terminus with the epitope Myc-DDK (Flag), under the control of a CMV promoter (mTL1A-Flag vector), was obtained from Origene Technologies (# MR203224).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy, Two Tailed Test

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: Two days post-confluent MEFs or 3T3-L1 cells were cultured in adipogenic induction medium supplemented with TL1A recombinant protein at the specified concentrations (0, 10, 20, 50, 100, 200 ng/mL). (A) Oil Red O staining was conducted after adipogenic induction for 7 days. (B) The average optical density (OD) was measured using a Universal Microplate Spectrophotometer at 490 nm. (C) Measurement of triglyceride (TG) content in MEFs 7 days after adipogenic differentiation. (D-E) Both Oil Red O staining and triglyceride assays in 3T3-L1 cells were performed on day 7 of differentiation. (F-G) qRT-PCR analysis was conducted to evaluate the expression of adipogenic markers aP2, Plin1, Plin2 and PPARγ in 3T3-L1 cells 7 days after adipogenic differentiation. (H) qRT-PCR analysis was conducted to evaluate the expression of VEGFA and several adipogenic markers C/EBPα/β/δ, PPARγ2, aP2, CD36, Plin1, Plin2, adispin, Glut4 and LPL in MEFs 7 days after adipogenic differentiation. * P < 0.05, ** P < 0.01, *** P < 0.001 vs . the group of adipocytes without TL1A treatment (n = 3).

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Cell Culture, Recombinant, Staining, Spectrophotometry, Quantitative RT-PCR, Expressing

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: (A-D) MEFs or 3T3-L1 cells were induced to undergo adipocyte differentiation for 3 days in the presence of the specified concentrations of TL1A. Total protein was extracted to determine C/EBPα, C/EBPβ, PPARγ2, PPARγ1 and aP2 protein expression by Western blotting with quantitation of band density. (E-H) MEFs or 3T3-L1 cells were induced to undergo adipocyte differentiation for 7 days with the indicated concentrations of TL1A. Total protein was extracted to determine PPARγ2, PPARγ1, CD36 and aP2 protein expression by Western blottingwith quantitation of band density. * P < 0.05, ** P < 0.01, *** P < 0.001 vs . the group of adipocytes without TL1A treatment (n = 3).

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Expressing, Western Blot, Quantitation Assay

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: After two days post-confluence, MEFs were treated with standard differentiation medium and TL1A recombinant protein (0, 10, 20, 50, 100, 200 ng/mL). At specified time points, cells were harvested for RNA isolation and subsequent qRT-PCR analysis. * P < 0.05 vs . the group of adipocytes without TL1A treatment.

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Recombinant, Isolation, Quantitative RT-PCR

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: MEFs were treated with standard differentiation medium and TL1A at the specified concentrations (0, 10, 20, 50, 100, 200 ng/mL) for 7 days. (A) Total cellular proteins were extracted, and the expression levels of β-catenin, phosphorylated YAP1 S127 (p-YAP1 S127 ) and total YAP1 proteins were determined by Western blotting. (B) The relative amounts of β-catenin protein expression were calculated according to the grayscale values and were showed in the histogram. (C) The quantitation of ratio (p-YAP1 S127 /total YAP1) to reflect the inactivation of YAP1 signaling pathway. (D-E) At the end of adipogenesis induction, protein expression of phosphorylated β-catenin (p- β-catenin) and YAP1 in the cytoplasm extract from MEFs was determined by Western blotting. (F-G) Nuclear extracts were isolated from the MEFs and analyzed for protein expression by Western blotting. Nuclear proteins were isolated to evaluated the expression of β-catenin, YAP1 and PPARγ proteins through Western blotting, accompanied by a quantitative analysis of band density. GAPDH and Lamin A/C served as the internal controls for cytoplasmic and nuclear extract, respectively. All the histograms represent the relative expression levels of proteins normalized by GAPDH or Lamin A/C. * P <0.05, ** P <0.01, *** P <0.001 vs . the group of MEFs without TL1A treatment.

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Expressing, Western Blot, Quantitation Assay, Isolation

Journal: PLOS One

Article Title: TL1A serves as a positive regulator to promote adipocyte differentiation

doi: 10.1371/journal.pone.0343036

Figure Lengend Snippet: Exogenous TL1A triggers the phosphorylation of YAP1 at Ser127, resulting in its cytoplasmic retention and functional inactivation. This event subsequently reduces β-catenin stability and prevents its nuclear translocation, thereby triggering the expression of essential adipogenic regulators and lipid metabolism-related proteins. Additionally, TL1A-mediated regulation of ABCA1 and ABCG1 expression facilitates cholesterol homeostasis, providing essential substrates for lipid droplet biogenesis. Collectively, these molecular mechanisms orchestrate the differentiation of MEFs and 3T3-L1 preadipocytes into terminally differentiated adipocytes.

Article Snippet: Human TL1A recombinant protein (Cat# 1319-TL-010/CF) was purchased from R&D Systems.

Techniques: Phospho-proteomics, Functional Assay, Translocation Assay, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: TNFSF15 treatment of human MDMs increases bacterial uptake and intracellular bacterial clearance. ( A ) MDMs were left untreated or treated with 10 ng/mL TNFSF15 for 48 hours ± pretreatment for 1 hour with neutralizing anti-DR3 antibodies (or isotype control) (n = 12 donors from 2 independent experiments, repeated in an additional 16 donors in the absence of neutralizing antibodies), and then co-cultured with E faecalis , AIEC, or S Typhimurium and assessed for intracellular bacterial clearance as per the Materials and Methods section. Mean colony forming units (CFU). ( B ) Human MDMs were left untreated or treated with 10 ng/mL TNFSF15 for 48 hours and then co-cultured with S Typhimurium-GFP or E coli –fluorescein isothiocyanate (FITC) bioparticles and uptake was assessed 20 minutes later by flow cytometry. Left : Representative flow cytometry with mean fluorescence intensity (MFI). Right : Summary graph of MFI (n = 8 donors from 2 independent experiments). ( C ) Human MDMs were left untreated or treated with 10 ng/mL TNFSF15 for 20 minutes. PDK1 activation was assessed by flow cytometry. Left : Representative flow cytometry. Right : Summary graph of fold PDK1 activation (n = 4 donors, similar results in an additional n = 4 over a time course). ( D–F ) Human MDMs were left untreated or treated with 10 ng/mL TNFSF15 ± GSK 233470 (PDK1 inhibitor; 1-hour pretreatment). ( D ) Fold PDK1 activation at 20 minutes (n = 4). ( E ) After 48 hours, cells were co-cultured with S Typhimurium–GFP or E coli –FITC bioparticles and uptake was assessed 20 minutes later (MFI) (n = 8 from 2 independent experiments). ( F ) Cell death was assessed at 48 hours by annexin V staining (n = 4). UV stimulation at 50–100 J/m 2 was used as a positive control. Means + SEM. Significance comparison is between inhibitor to the vehicle control for the corresponding TNFSF15 treatment condition for panels D–E . ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 × 10 -5 . NT, no treatment; Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Control, Cell Culture, Flow Cytometry, Fluorescence, Activation Assay, Staining, Positive Control, Comparison

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: TNFSF15 promotes ROS and RNS pathways in MDMs. ( A , B , and G ) MDMs were treated with 10 ng/mL TNFSF15 for 48 hours. ( A ) ROS induction (n = 6; similar results in an additional n = 4). ( B ) NADPH oxidase members (n = 6; similar results additional n = 6). ( G ) NOS2 expression (n = 6; similar results in an additional n = 6). ( C–E , H , and I ) MDMs were transfected with scrambled or the indicated siRNAs, and then treated with 10 ng/mL TNFSF15 for 48 hours. ( C and H ) Expression of the indicated proteins by flow cytometry (n = 6). ( D ) ROS production (n = 6). ( E and I ) Intracellular bacterial clearance (n = 10 from 2 independent experiments). ( F ) MDMs were transfected with scrambled or the indicated siRNAs and cell death was assessed by annexin V staining (n = 4). UV stimulation at 50–100 J/m 2 was used as a positive control for cell death. Means + SEM. ( A–C , G , and H ) Representative flow cytometry with MFI values is shown. ( D , E , and I ) Significance is between scrambled and the target siRNA for TNFSF15-treated cells. ∗∗ P < .01; ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 × 10 -5 . MFI, mean fluorescence intensity; NT, no treatment; scr, scrambled; Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Expressing, Transfection, Flow Cytometry, Staining, Positive Control, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: TNFSF15 promotes autophagy pathways in MDMs. ( A and B ) MDMs were treated with 10 ng/mL TNFSF15 for 48 hours. ( A ) LC3II expression (n = 6; similar results in an additional n = 4). ( B ) Autophagy molecule expression (n = 6; similar results in an additional n = 8 for ATG16L1 and n = 4 for ATG5). ( C–F ) MDMs were transfected with scrambled or the indicated siRNAs. ( C ) Expression of the indicated proteins by flow cytometry (n = 6). ( D and E ) Cells then were treated with 10 ng/mL TNFSF15 for 48 hours. ( D ) LC3II expression (n = 6). ( E ) Intracellular bacterial clearance (n = 10 from 2 independent experiments). ( F ) Cell death was assessed by annexin V staining (n = 4). UV stimulation at 50–100 J/m 2 was used as a positive control. Means + SEM. ( A–C ) Representative flow cytometry with mean fluorescence intensity (MFI) values shown. Significance is between scrambled and the target siRNA for TNFSF15-treated cells for panels D and E . ∗∗∗ P < .001; † P <1 × 10 -4 ; †† P < 1 × 10 -5 . NT, no treatment; scr, scrambled; Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Expressing, Transfection, Flow Cytometry, Staining, Positive Control, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: TNFSF15-induced ROS, RNS, and autophagy pathways cooperate to mediate intracellular bacterial clearance. MDMs were transfected with scrambled siRNA or the indicated siRNA, alone or in combination (comb). ( A ) Cells then were treated with TNFSF15 for 48 hours. Intracellular bacterial clearance was assessed (colony-forming units [CFU]) (n = 8 from 2 independent experiments). Significance is between scrambled and the target siRNA for TNFSF15-treated cells. ( B ) Cell death was assessed by annexin V staining (n = 4). UV stimulation at 50–100 J/m 2 was used as a positive control. Mean + SEM. ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 × 10 -5 . scr, scrambled; Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Transfection, Staining, Positive Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: The TRAF2/RIP1/RIP3 pathway is required for TNFSF15-induced bacterial uptake and intracellular bacterial clearance pathways. ( A ) Model of proposed TNFSF15–DR3–initiated signaling in MDMs. ( B–J ) MDMs were transfected with siRNA to TRADD or to FADD, MALT1, caspase-8 alone or in combination (F/M/C), or to TRAF2, RIP1, or RIP3 alone or in combination (T/R1/R3). ( B ) Fold mRNA expression (n = 6). ( C–H ) Cells then were treated with 10 ng/mL TNFSF15. ( C ) Fold PDK1 activation at 20 minutes (n = 6 donors, similar results in n = 4 for single-siRNA–transfected groups). ( D ) Forty-eight hours later bacterial uptake was assessed (n = 8 from 2 independent experiments). ( E ) Fold cleaved (clv) caspase-8 or cleaved (clv) IL1β at 20 minutes. Left : Representative flow cytometry with MFI values. Right : Summary of fold cleaved caspase-8 or cleaved IL1β (n = 8 from 2 independent experiments). ( F ) Cytokine secretion at 24 hours (n = 6). ( G ) ROS, NOS2, or LC3II expression at 48 hours (n = 12 from 3 independent experiments). ( H ) After 48 hours cells were assessed for intracellular bacterial clearance (colony-forming units [CFU]) (n = 8 from 2 independent experiments). ( I and J ) Cell death was assessed by annexin V staining (n = 4). UV stimulation at 50–100 J/m 2 was used as a positive control. Means + SEM. ( C–H ) Significance is between scrambled and the target siRNA for the corresponding TNFSF15-treated conditions or as indicated. ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 × 10 -5 . MFI, mean fluorescence intensity; NT, no treatment; scr, scrambled; Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Transfection, Expressing, Activation Assay, Flow Cytometry, Staining, Positive Control, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: MAPK and NF-κB signaling is required for TNFSF15-induced microbial clearance. ( A ) MDMs were treated with 10 ng/mL TNFSF15 and assessed for activation of the indicated phospho-proteins at 15 minutes. Left : Representative flow cytometry with mean fluorescence intensity (MFI) values shown. Right : Summary graph of fold phospho-protein activation (n = 6). ( B–E ) MDMs were transfected with scrambled, ERK/p38/JNK (MAPK), or NF-κB essential modulator (NEMO) (NF-κB pathway), alone or in combination (comb). ( B ) Expression of the indicated proteins by flow cytometry (n = 6, similar results in an additional n = 4 for MAPKs). ( C and D ) Cells then were treated with 10 ng/mL TNFSF15 for 48 hours. ( C ) ROS, NOS2, or LC3II expression (n = 4, similar results in additional n = 8). ( D ) Intracellular bacterial clearance (colony-forming units [CFU]) (n = 4, similar results in an additional n = 8). ( E ) Cell death was assessed by annexin V staining (n = 4, similar results in an additional n = 4). UV stimulation at 50–100 J/m 2 was used as a positive control. Means + SEM. ∗∗ P < .01; ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 × 10 -5 . NT, no treatment; scr, scrambled; Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Activation Assay, Flow Cytometry, Fluorescence, Transfection, Expressing, Staining, Positive Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: The TRAF2/RIP1/RIP3 pathway is required for optimal TNFSF15-induced MAPK and NF-κB signaling. ( A and B ) MDMs were transfected with scrambled, a combination of ERK/p38/JNK (MAPKs), or NEMO siRNA, and then treated with 10 ng/mL TNFSF15. Fold mRNA expression at 4 and 48 hours (n = 6, similar results in an additional n = 6). ( C and D ) MDMs were transfected with scrambled, TRADD, combined FADD/MALT1/caspase-8 (F/M/C), or combined TRAF2/RIP1/RIP3 (T/R1/R3) siRNA, and then treated with 10 ng/mL TNFSF15. ( C ) Fold phospho-protein expression at 15 minutes (n = 8 from 2 independent experiments). ( D ) Fold phospho-protein expression at the indicated times (n = 8 from 2 independent experiments). Means + SEM. Significance is between scrambled and the target siRNA for the corresponding TNFSF15-treated cells or as indicated. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 ×10 -5 . Scr, scrambled; Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Transfection, Expressing

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: Autocrine/paracrine TNFSF15 is required for optimal levels of NOD2-induced intracellular bacterial clearance. ( A ) MDMs were treated with 100 μg/mL MDP for 48 hours ± neutralizing αDR3 antibodies (or isotype control) (1 hour pretreatment). Intracellular bacterial clearance was assessed (colony-forming units [CFU]) (n = 12 from 2 independent experiments; similar results in an additional n = 4). ( B–F ) MDMs were transfected with scrambled or ( B , C , and F ) DR3 siRNA or ( D–F ) TNFSF15 siRNA. ( B and D ) Expression of the indicated proteins by flow cytometry (n = 8 from 2 independent experiments for DR3; n = 4 for TNFSF15). ( C and E ) Cells then were treated with 100 μg/mL MDP for 48 hours and intracellular bacterial clearance was assessed (n = 12 from 2 independent experiments). ( F ) Cell death was assessed by annexin V staining (n = 4). UV stimulation at 50–100 J/m 2 was used as a positive control. ( G – J ) MDMs were treated with 100 μg/mL MDP for 48 hours ± TAPI-1 (inhibits TACE; 1 hour pretreatment) ± 10 ng/mL TNFSF15. ( G ) TNFSF15 secretion (n = 4). ( H ) Cell surface TNFSF15 by flow cytometry with representative flow cytometry and summary graph of mean fluorescence intensity (MFI) (n = 6; similar results in an additional n = 4). ( I ) Intracellular bacterial clearance was assessed (CFU) (n = 8 from 2 independent experiments for E faecalis and AIEC with similar results in an additional n = 4; n = 4 for S Typhimurium with similar results for an additional n = 4). ( J ) Cell death was assessed by annexin V staining (n = 4). UV stimulation at 50–100 J/m 2 was used as a positive control. Means + SEM. Significance is between isotype and the indicated antibody, scrambled and the target siRNA, or vehicle and inhibitor for the corresponding MDP-treated condition or as indicated. ∗∗ P < .01; ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 × 10 -5 . NT, no treatment; scr, scrambled; Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Control, Transfection, Expressing, Flow Cytometry, Staining, Positive Control, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: Autocrine/paracrine TNFSF15 promotes NOD2-induced bacterial uptake. ( A and B ) MDMs were treated with 100 μg/mL MDP ± neutralizing αDR3 antibodies (or isotype control). ( A ) Fold PDK1 activation at 20 minutes (n = 4). ( B ) After 48 hours, bacterial uptake was assessed (n = 8 from 2 independent experiments). ( C and D ) MDMs were treated with 100 μg/mL MDP ± neutralizing αDR3 antibodies ± PS48 (activates PDK1). ( C ) Fold PDK1 activation at 20 minutes (n = 6; similar results in an additional n = 4). ( D ) After 48 hours bacterial uptake was assessed (n = 10 from 2 independent experiments). PS48 alone is shown as a control. ( E and F ) MDMs were treated with 100 μg/mL MDP ± TACE ± 10 ng/mL TNFSF15. ( E ) Fold PDK1 activation at 20 minutes (n = 6). ( F ) After 48 hours bacterial uptake was assessed (n = 8 from 2 independent experiments). Means + SEM. ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 × 10 -5 . FITC, fluorescein isothiocyanate; MFI, mean fluorescence intensity; Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Control, Activation Assay, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: Autocrine/paracrine TNFSF15 promotes NOD2-induced antimicrobial pathways. ( A–E ) MDMs were treated with 100 μg/mL MDP for 48 hours ± neutralizing αDR3 antibodies (or isotype control). ( A ) ROS induction (n = 6; similar results in an additional n = 4). ( B–E ) Expression of the indicated proteins (n = 6; similar results in an additional n = 6). ( F–I ) MDMs were transfected with empty vector or vectors expressing p47phox, NOS2, ATG5, alone or in combination, and treated with 100 μg/mL MDP for 48 hours ± neutralizing αDR3 antibodies (1 hour pretreatment). ( F ) p47phox expression and ROS production. ( G ) NOS2 expression. ( H ) ATG5 and LC3II expression (n = 4, similar results in an additional n = 4). ( I ) Intracellular bacterial clearance (n = 8 from 2 independent experiments). Means + SEM. ( A–E ) Significance is between isotype control and neutralizing αDR3 antibodies for MDP-treated cells. ∗∗ P < .01; ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 × 10 -5 . CFU, colony-forming unit; MFI, mean fluorescence intensity; Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Control, Expressing, Transfection, Plasmid Preparation, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: Autocrine/paracrine TNFSF15-initiated MAPK and NF-κB activation promotes NOD2-induced antimicrobial pathways. ( A and B ) MDMs were treated with 100 μg/mL MDP ± neutralizing αDR3 antibodies (1 hour pretreatment) (or isotype control). Fold phospho-protein expression (n = 8 from 2 independent experiments). ( C–F ) MDMs were transfected with empty vector or vectors leading to constitutively active ERK, p38, or JNK (ca-MAPK when in combination) or NF-κB (ca-NF-κB), alone or in combination (comb), and treated with 100 μg/mL MDP for 48 hours ± neutralizing αDR3 antibodies. ( C and F ) Fold phospho-protein expression (n = 4). ( D ) ROS production and NOS2 or LC3II expression (mean fluorescence intensity [MFI]) (n = 4, similar results in an additional n = 4). ( E ) Intracellular bacterial clearance (colony-forming unit [CFU]) (n = 8 from 2 independent experiments for E faecalis and S Typhimurium; n = 4 with similar results in an additional n = 4 for AIEC). Means + SEM. ( A and B ) Significance is between isotype control to neutralizing αDR3 antibodies for MDP-treated cells or ( C–F ) empty vector–transfected cells to target vector-transfected cells for αDR3-blocked, MDP-treated cells. ∗∗ P < .01; ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 × 10 -5 . Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Activation Assay, Control, Expressing, Transfection, Plasmid Preparation, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: TNFSF15 signaling potentiates NOD2-mediated intracellular bacterial clearance and autocrine/paracrine TNFSF15 promotes bacterial clearance upon stimulation of multiple PRRs. ( A and B ) MDMs were pretreated with 100 μg/mL MDP and 24 hours later treated with 10 ng/mL TNFSF15 for an additional 48 hours. ( A ) Timeline schematic for treatment strategy. ( B ) Intracellular bacterial clearance was assessed (colony-forming units [CFU]) (n = 12 from 2 independent experiments). ( C ) MDMs were treated with 10 μg/mL Pam3Cys, 0.1 μg/mL lipid A, or 10 μg/mL CpG for 48 hours ± neutralizing αDR3 antibodies (1 hour pretreatment) (or isotype control), then co-cultured with the indicated bacteria, and intracellular bacterial clearance was assessed (colony-forming units [CFU]) (n = 12 from 2 independent experiments). Significance is between isotype control to neutralizing αDR3 antibodies for the corresponding treatment condition or as indicated. Means + SEM. † P < 1 × 10 -4 ; †† P < 1 × 10 -5 .

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Control, Cell Culture, Bacteria

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: Autocrine/paracrine TNFSF15 promotes intracellular bacterial clearance in mouse BMDMs and intestinal myeloid cells. ( A and B ) Mouse BMDMs were treated with 10 ng/mL TNFSF15 or 0.1 μg/mL lipid A ± neutralizing αTNFSF15 antibodies (30 minutes pretreatment) (or isotype control). ( A ) Cytokine secretion after 24 hours (8 replicates, representative of 2 independent experiments). ( B ) Forty-eight hours later intracellular bacterial clearance was assessed (colony-forming units [CFU]) (10 replicates from 2 independent experiments). ( C–E ) Colonic lamina propria (LP) CD11b + myeloid cells were isolated. ( C ) Representative flow cytometry showing purity of intestinal myeloid cells (percentages indicated). CD19 + and CD3 + cells are minimal, with mesenteric lymph node (MLN) shown as a positive control for these markers. ( D and E ) Cells were co-cultured with S Typhimurium ± αTNFSF15 antibodies (30 minutes pretreatment) (or isotype control) (n = 7 from 2 independent experiments). ( D ) Cytokine secretion at 12 hours. ( E ) Left : CFU were assessed after 20 minutes. Right : Gentamicin was added and cells were cultured for an additional 220 minutes (total of 4 hours) (CFU). Significance is between isotype control to neutralizing αTNFSF15 antibodies for the corresponding treatment condition or as indicated. Means + SEM. ∗∗ P < .01; ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 × 10 -5 . Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Control, Isolation, Flow Cytometry, Positive Control, Cell Culture

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: MDMs from high TNFSF15-expressing rs6478108 TT IBD risk carriers show increased NOD2-initiated antimicrobial pathways. MDMs from rs6478108 TT, TC, or CC carriers were left untreated or treated with 100 μg/mL MDP. ( A ) TNFSF15 surface expression by flow cytometry at 24 hours (n = 10 donors/genotype). ( B ) TNFSF15 secretion at 24 hours (n = 10/genotype). ( C ) Fold PDK1 activation at 20 minutes (n = 10/genotype). ( D ) Bacterial uptake at 20 minutes (n = 10/genotype). ( E ) p47phox expression and ROS production at 48 hours (n = 10/genotype). ( F ) NOS2 expression at 48 hours (n = 10/genotype). ( G ) ATG5 and LC3II expression at 48 hours (n = 10/genotype). ( H ) After 48 hours intracellular bacterial clearance was assessed (colony-forming units [CFU]) (n = 10/genotype). Means + SEM. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 × 10 -5 . MFI, mean fluorescence intensity; Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Expressing, Flow Cytometry, Activation Assay, Fluorescence

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: MDMs from high TNFSF15-expressing rs6478108 TT IBD risk carriers show increased DR3-initiated antimicrobial pathways. MDMs from rs6478108 TT, TC, or CC carriers were left untreated or treated with 10 ng/mL TNFSF15. ( A ) Fold PDK1 activation at 20 minutes (n = 10 donors/genotype). ( B ) Bacterial uptake at 20 minutes (n = 10/genotype). ( C ) p47 expression and ROS production at 48 hours (n = 10/genotype). ( D ) NOS2 expression at 48 hours (n = 10/genotype). ( E ) ATG5 and LC3II expression at 48 hours (n = 10/genotype). ( F ) After 48 hours intracellular bacterial clearance was assessed (colony-forming units [CFU]) (n = 10/genotype). Means + SEM. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; † P < 1 × 10 -4 ; †† P < 1 × 10 -5 . Tx, treatment.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Expressing, Activation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants

doi: 10.1016/j.jcmgh.2020.08.003

Figure Lengend Snippet: Model of TNFSF15 regulation of antimicrobial pathways. Upon PRR stimulation of macrophages, TACE is activated and leads to the release of soluble TNFSF15. Soluble TNFSF15 then feeds back to interact with DR3 on macrophages to initiate TRAF2/RIP1/RIP3 and FADD/MALT1/caspase-8 signaling pathways. TRAF2/RIP1/RIP3 pathway activation leads to PDK1-dependent bacterial uptake and MAPK- and NF-κB–dependent up-regulation of ROS (p40phox, p47phox, p67phox), RNS, and autophagy (ATG5, ATG16L1) pathways, which promote intracellular bacterial clearance. MDMs from high TNFSF15-expressing rs6478108 TT IBD risk carriers in the TNFSF15 region show increased efficacy in inducing these antimicrobial pathways, and, conversely, MDMs from low TNFSF15-expressing rs6478108 CC IBD risk carriers clear bacteria less effectively.

Article Snippet: In some cases, cells were treated first with neutralizing anti-TNFSF15 antibodies (2 μg/mL, 5G4.6; Bio X Cell, Lebanon, NH) for 30 minutes.

Techniques: Protein-Protein interactions, Activation Assay, Expressing, Bacteria