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: 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: Scientific Reports

Article Title: Direct signaling of TL1A-DR3 on fibroblasts induces intestinal fibrosis in vivo

doi: 10.1038/s41598-020-75168-5

Figure Lengend Snippet: Fibroblast-selective DR3 deficiency results in severe disease activity: ( A – D ) Percent body weight loss, stool consistency, stool blood, and composite disease activity index are shown for the adoptive transfer model of colitis, at indicated time points for transfers of naïve Tl1a-Tg T cells into Rag −/− , Rag −/− Dr3 −/− , or Rag −/− Dr3 ∆Col1a2 mice. Data are represented as means ± SEM; * indicates p < 0.05, ** p < 0.01 Rag −/− Dr3 −/− vs Rag −/− ; # indicates p < 0.05 Rag −/− Dr3 −/− vs Rag −/− Dr3 ∆Col1a2 ; n = 9–10 mice per group. ( E – F ) Representative gross colonic specimens, quantitated macroscopic pathology scores, and colon lengths; representative H&E cecal sections and quantitated histopathological scores are shown; * indicates p < 0.05, ** p < 0.01. Pooled data of 3 independent experiments are represented.

Article Snippet: The plate was then incubated at 37 °C and 5% CO 2 for 1 h. After collagen polymerization, 1.0 mL of culture medium, with/without mouse recombinant Tl1a (R&D Systems, Minneapolis, MN) at a concentration 100 ng/mL, or ROCK inhibitor Y27632 (Stemcell Technologies, Cambridge, MA) at a concentration 25 μM , was added to the top of each collagen gel lattice.

Techniques: Activity Assay, Adoptive Transfer Assay

Journal: Scientific Reports

Article Title: Direct signaling of TL1A-DR3 on fibroblasts induces intestinal fibrosis in vivo

doi: 10.1038/s41598-020-75168-5

Figure Lengend Snippet: Transcriptomic analysis of Tl1a-stimulated intestinal fibroblasts identifies cytoskeletal contraction-related genes and the Rho signaling pathway: ( A ) Heatmap generated from transcriptome analysis of isolated cecal fibroblasts from three individual mice shown as untreated (a, b, c) and Tl1a-treated (a + Tl1a, etc.) pairs; BRB array tools, (brb.nci.nih.gov/BRB-ArrayTools). ( B ) Independent validation cohort was used to confirm several genes in the index group; matched pairs displayed as untreated (ut) or TL1A-treated fibroblasts with direct increase in expression for each sample shown, * indicates p < 0.05. ( C ) Genetic ontology pathway enrichment of differentially expressed genes induced by TL1A in ( A ).

Article Snippet: The plate was then incubated at 37 °C and 5% CO 2 for 1 h. After collagen polymerization, 1.0 mL of culture medium, with/without mouse recombinant Tl1a (R&D Systems, Minneapolis, MN) at a concentration 100 ng/mL, or ROCK inhibitor Y27632 (Stemcell Technologies, Cambridge, MA) at a concentration 25 μM , was added to the top of each collagen gel lattice.

Techniques: Generated, Isolation, Biomarker Discovery, Expressing

Journal: Scientific Reports

Article Title: Direct signaling of TL1A-DR3 on fibroblasts induces intestinal fibrosis in vivo

doi: 10.1038/s41598-020-75168-5

Figure Lengend Snippet: Inhibition of Rho kinase pathway modulates morphology and function of intestinal fibroblasts: ( A ) Gap closure assay and morphology of migrating intestinal fibroblasts isolated from three individual Tl1a-Tg mice, in the presence (or absence) of 25uM ROCK inhibitor Y27632, shown as treated and un-treated pairs. Blue arrow denotes direction of migration with solid vs dotted line highlighting cell extensions at migrating front; upper panels 100× objective, lower panels 200× objective. ( B ) Collagen contraction assay of intestinal fibroblasts isolated and treated as in ( A ). ( C ) Collagen contraction assay of WT or DR3 − / − intestinal fibroblasts treated with 100 ng/mL mouse recombinant Tl1a, 25uM ROCK inhibitor Y27632, or both. Data normalized to genotype control with bars representative of means; * indicates p < 0.05, ** p < 0.01.

Article Snippet: The plate was then incubated at 37 °C and 5% CO 2 for 1 h. After collagen polymerization, 1.0 mL of culture medium, with/without mouse recombinant Tl1a (R&D Systems, Minneapolis, MN) at a concentration 100 ng/mL, or ROCK inhibitor Y27632 (Stemcell Technologies, Cambridge, MA) at a concentration 25 μM , was added to the top of each collagen gel lattice.

Techniques: Inhibition, Isolation, Migration, Contraction Assay, Recombinant, Control