Journal: Research

Article Title: Integrated Single-Cell Profiling Reveals TL1A as a Biomarker and Driver of Type 2 Inflammation via Macrophage-Dependent Immunoregulation in Asthma

doi: 10.34133/research.1190

Figure Lengend Snippet: Tumor necrosis factor-like ligand 1A (TL1A) expression and secretion increase as asthma progresses. (A) Western blot analysis of TL1A protein expression in the lung tissues in the wild-type and ovalbumin (OVA)-induced model groups. (B) Changes in the cytokine concentrations of TL1A and immunoglobulin E (IgE) in serum over time following the final challenge in cases and controls. (C) Changes in the cytokine concentrations of TL1A, interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 13 (IL-13), and interferon gamma (IFN-γ) in bronchoalveolar lavage fluid (BAL) over time following the final challenge in cases and controls. (D) Schematic summarizing the timeline of the in vivo asthma modeling with different allergen exposure frequencies. (E) Comparison of total cell counts in the BAL in each group for different challenge numbers. (F) Comparison of IL-13 concentrations in BAL changed for different challenge numbers. (G) Representative plots showing changes in eosinophil counts for different challenge numbers. (H) Changes in IgE concentrations in blood samples for different challenge numbers. (I to K) Representative plots showing Masson staining, hematoxylin and eosin (HE) staining, and periodic acid–Schiff (PAS) staining of pulmonary airway tissue in mice for different challenge numbers. (L) Representative plots showing comparison of S100A4 + CD8 + effector memory T (Tem) cell counts for different challenge numbers. (M) Comparison of TL1A expression level in lung tissue samples from each group. (N and O) Comparison of TL1A concentrations in serum/BAL samples in each group. Data are presented as mean ± SD (error bars) from at least 3 independent experiments, with n = 6 mice per group per experiment (A and E to O). Time-course studies (B and C) used n = 6 mice per time point. * P < 0.05 and ** P < 0.01 compared with the respective control groups. Histological scoring and flow cytometry analysis were performed by investigators blinded to the experimental groups.

Article Snippet: The ELISA kits used were as follows: mouse CCL8 (ZCI BIO), mouse CXCL-2 (MultiSciences), mouse CXCL-10 (MultiSciences), mouse IFN-γ (MultiSciences), mouse IL-4 (MultiSciences), mouse IL-6 (MultiSciences), mouse IL-13 (MultiSciences), IFN-γ (MultiSciences), mouse total IgE (Jianglaibio), mouse TL1A (ZCI BIO), and human TL1A (ZCI BIO, Catalog No. ZC-35762, had a stated lower limit of detection of 5 pg/ml, as per the manufacturer’s specifications).

Techniques: Expressing, Western Blot, In Vivo, Comparison, Staining, Control, Flow Cytometry

Journal: Research

Article Title: Integrated Single-Cell Profiling Reveals TL1A as a Biomarker and Driver of Type 2 Inflammation via Macrophage-Dependent Immunoregulation in Asthma

doi: 10.34133/research.1190

Figure Lengend Snippet: Myeloid-cell-specific Tnfsf15 -knockout resulted in the attenuation of allergic airway inflammation. (A) Myeloid-cell-specific Tnfsf15 -knockout strategy. (B) Genotyping was confirmed using tail DNA genomic polymerase chain reaction. (C) Immunomagnetic bead sorting was used to isolate F4/80 + cells, and the expression level of tumor necrosis factor-like ligand 1A (TL1A) was quantified using Western blotting. (D to G) Representative plots showing the proportions of 4 major types of immune cells (macrophages, CD8 + T cells, CD4 + T cells, and B cells) infiltrating the lung tissues in each group. (H) Representative hematoxylin and eosin (HE) staining among the different groups and quantification of the airway inflammation score. Black scale bar, 50 µm. (I) Representative periodic acid–Schiff (PAS) staining among the different groups and quantification of the airway mucus score. (J) Representative Masson’s trichrome staining among the different groups and quantification of the collagen volume fraction. (K and L) Lung eosinophil or neutrophil counts, as determined by flow cytometry. (M) Total cell counts in bronchoalveolar lavage fluid (BAL). (N) Concentrations of interleukin 4 (IL-4) in BAL. (O) Concentrations of interleukin 13 (IL-13) in BAL. Data are presented as mean ± SD. Experiments were repeated 3 times with n = 6 to 8 mice per group per experiment. Cell sorting and Western blot (C) used pooled cells from n = 4 mice. Flow cytometry analyses (D to G and K) and histological scoring (H to J) were performed by investigators blinded to genotype and treatment. * P < 0.05 and ** P < 0.01 compared with the respective groups.

Article Snippet: The ELISA kits used were as follows: mouse CCL8 (ZCI BIO), mouse CXCL-2 (MultiSciences), mouse CXCL-10 (MultiSciences), mouse IFN-γ (MultiSciences), mouse IL-4 (MultiSciences), mouse IL-6 (MultiSciences), mouse IL-13 (MultiSciences), IFN-γ (MultiSciences), mouse total IgE (Jianglaibio), mouse TL1A (ZCI BIO), and human TL1A (ZCI BIO, Catalog No. ZC-35762, had a stated lower limit of detection of 5 pg/ml, as per the manufacturer’s specifications).

Techniques: Knock-Out, Polymerase Chain Reaction, Expressing, Western Blot, Staining, Flow Cytometry, FACS

Journal: Research

Article Title: Integrated Single-Cell Profiling Reveals TL1A as a Biomarker and Driver of Type 2 Inflammation via Macrophage-Dependent Immunoregulation in Asthma

doi: 10.34133/research.1190

Figure Lengend Snippet: Identification of C-C motif chemokine ligand 8 (CCL8) as the key link in the tumor necrosis factor-like ligand 1A (TL1A)-induced allergic inflammatory response. (A) Heatmap showing changes in proallergic chemokine expression levels after gene knockdown. (B) Relative gene expression of CCL8 in lung tissue, quantified using polymerase chain reaction (PCR). (C) Relative gene expression of CCL8 in sorted F4/80 + cell groups in lung tissue, quantified using PCR. (D) CCL8 concentration in bronchoalveolar lavage fluid (BAL) measured using enzyme-linked immunosorbent assay (ELISA). (E) Relative gene expression of CCL8 in lung tissue, quantified using PCR in Tnfsf15 -knockout (KO) mice. (F) CCL8 concentration in BAL measured using ELISA in Tnfsf15 -KO mice. (G) CCL8 expression in the lung tissue of Tnfsf15 transgenic and nontransgenic mice. (H) The correlation between TL1A and CCL8 expression in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort (based on GSE76262 ). (I) Schematic of the murine ovalbumin-induced asthma models following recombinant TL1A or anti-C-C motif chemokine receptor 8 (anti-CCR8) intervention. (J) Hematoxylin and eosin (HE) staining of lung tissue after allergen challenge and recombinant TL1A (rTL1A) or anti-CCR8 interventions. Black scale bar, 50 µm. (K) Total cell numbers in BAL samples. (L to N) Concentrations of CCL8, interleukin 4 (IL-4), and interleukin 13 (IL-13) in BAL samples from the different groups. Data are presented as mean ± SD from at least 2 independent experiments, with n = 5 to 6 mice per group per experiment. PCR, ELISA, and histological assessments were conducted by researchers blinded to treatment groups. Public dataset correlation (H) used n = samples from the GSE76262 dataset. * P < 0.05 and ** P < 0.01 compared with the respective groups.

Article Snippet: The ELISA kits used were as follows: mouse CCL8 (ZCI BIO), mouse CXCL-2 (MultiSciences), mouse CXCL-10 (MultiSciences), mouse IFN-γ (MultiSciences), mouse IL-4 (MultiSciences), mouse IL-6 (MultiSciences), mouse IL-13 (MultiSciences), IFN-γ (MultiSciences), mouse total IgE (Jianglaibio), mouse TL1A (ZCI BIO), and human TL1A (ZCI BIO, Catalog No. ZC-35762, had a stated lower limit of detection of 5 pg/ml, as per the manufacturer’s specifications).

Techniques: Expressing, Knockdown, Gene Expression, Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Knock-Out, Transgenic Assay, Recombinant, Staining

Journal: Research

Article Title: Integrated Single-Cell Profiling Reveals TL1A as a Biomarker and Driver of Type 2 Inflammation via Macrophage-Dependent Immunoregulation in Asthma

doi: 10.34133/research.1190

Figure Lengend Snippet: Tumor necrosis factor-like ligand 1A (TL1A) expression and secretion increase as asthma progresses. (A) Western blot analysis of TL1A protein expression in the lung tissues in the wild-type and ovalbumin (OVA)-induced model groups. (B) Changes in the cytokine concentrations of TL1A and immunoglobulin E (IgE) in serum over time following the final challenge in cases and controls. (C) Changes in the cytokine concentrations of TL1A, interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 13 (IL-13), and interferon gamma (IFN-γ) in bronchoalveolar lavage fluid (BAL) over time following the final challenge in cases and controls. (D) Schematic summarizing the timeline of the in vivo asthma modeling with different allergen exposure frequencies. (E) Comparison of total cell counts in the BAL in each group for different challenge numbers. (F) Comparison of IL-13 concentrations in BAL changed for different challenge numbers. (G) Representative plots showing changes in eosinophil counts for different challenge numbers. (H) Changes in IgE concentrations in blood samples for different challenge numbers. (I to K) Representative plots showing Masson staining, hematoxylin and eosin (HE) staining, and periodic acid–Schiff (PAS) staining of pulmonary airway tissue in mice for different challenge numbers. (L) Representative plots showing comparison of S100A4 + CD8 + effector memory T (Tem) cell counts for different challenge numbers. (M) Comparison of TL1A expression level in lung tissue samples from each group. (N and O) Comparison of TL1A concentrations in serum/BAL samples in each group. Data are presented as mean ± SD (error bars) from at least 3 independent experiments, with n = 6 mice per group per experiment (A and E to O). Time-course studies (B and C) used n = 6 mice per time point. * P < 0.05 and ** P < 0.01 compared with the respective control groups. Histological scoring and flow cytometry analysis were performed by investigators blinded to the experimental groups.

Article Snippet: The recombinant proteins, small-molecular compounds, or inhibitors were purchased as indicated: recombinant mouse IL-13, IL-33, and TSLP protein (MCE); recombinant human IL-2, IL-4, TNF-α, and IFN-γ protein (Abbkine); poly(I:C) (Selleck); lipopolysaccharide (Sigma-Aldrich); IL-1β (Thermo Fisher); and small-molecule inhibitors BAY11-7082, LY294002, PD98059, SB203580, and SP600125 (Selleck).

Techniques: Expressing, Western Blot, In Vivo, Comparison, Staining, Control, Flow Cytometry

Journal: Research

Article Title: Integrated Single-Cell Profiling Reveals TL1A as a Biomarker and Driver of Type 2 Inflammation via Macrophage-Dependent Immunoregulation in Asthma

doi: 10.34133/research.1190

Figure Lengend Snippet: Myeloid-cell-specific Tnfsf15 -knockout resulted in the attenuation of allergic airway inflammation. (A) Myeloid-cell-specific Tnfsf15 -knockout strategy. (B) Genotyping was confirmed using tail DNA genomic polymerase chain reaction. (C) Immunomagnetic bead sorting was used to isolate F4/80 + cells, and the expression level of tumor necrosis factor-like ligand 1A (TL1A) was quantified using Western blotting. (D to G) Representative plots showing the proportions of 4 major types of immune cells (macrophages, CD8 + T cells, CD4 + T cells, and B cells) infiltrating the lung tissues in each group. (H) Representative hematoxylin and eosin (HE) staining among the different groups and quantification of the airway inflammation score. Black scale bar, 50 µm. (I) Representative periodic acid–Schiff (PAS) staining among the different groups and quantification of the airway mucus score. (J) Representative Masson’s trichrome staining among the different groups and quantification of the collagen volume fraction. (K and L) Lung eosinophil or neutrophil counts, as determined by flow cytometry. (M) Total cell counts in bronchoalveolar lavage fluid (BAL). (N) Concentrations of interleukin 4 (IL-4) in BAL. (O) Concentrations of interleukin 13 (IL-13) in BAL. Data are presented as mean ± SD. Experiments were repeated 3 times with n = 6 to 8 mice per group per experiment. Cell sorting and Western blot (C) used pooled cells from n = 4 mice. Flow cytometry analyses (D to G and K) and histological scoring (H to J) were performed by investigators blinded to genotype and treatment. * P < 0.05 and ** P < 0.01 compared with the respective groups.

Article Snippet: The recombinant proteins, small-molecular compounds, or inhibitors were purchased as indicated: recombinant mouse IL-13, IL-33, and TSLP protein (MCE); recombinant human IL-2, IL-4, TNF-α, and IFN-γ protein (Abbkine); poly(I:C) (Selleck); lipopolysaccharide (Sigma-Aldrich); IL-1β (Thermo Fisher); and small-molecule inhibitors BAY11-7082, LY294002, PD98059, SB203580, and SP600125 (Selleck).

Techniques: Knock-Out, Polymerase Chain Reaction, Expressing, Western Blot, Staining, Flow Cytometry, FACS

Journal: Research

Article Title: Integrated Single-Cell Profiling Reveals TL1A as a Biomarker and Driver of Type 2 Inflammation via Macrophage-Dependent Immunoregulation in Asthma

doi: 10.34133/research.1190

Figure Lengend Snippet: Identification of C-C motif chemokine ligand 8 (CCL8) as the key link in the tumor necrosis factor-like ligand 1A (TL1A)-induced allergic inflammatory response. (A) Heatmap showing changes in proallergic chemokine expression levels after gene knockdown. (B) Relative gene expression of CCL8 in lung tissue, quantified using polymerase chain reaction (PCR). (C) Relative gene expression of CCL8 in sorted F4/80 + cell groups in lung tissue, quantified using PCR. (D) CCL8 concentration in bronchoalveolar lavage fluid (BAL) measured using enzyme-linked immunosorbent assay (ELISA). (E) Relative gene expression of CCL8 in lung tissue, quantified using PCR in Tnfsf15 -knockout (KO) mice. (F) CCL8 concentration in BAL measured using ELISA in Tnfsf15 -KO mice. (G) CCL8 expression in the lung tissue of Tnfsf15 transgenic and nontransgenic mice. (H) The correlation between TL1A and CCL8 expression in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes (U-BIOPRED) cohort (based on GSE76262 ). (I) Schematic of the murine ovalbumin-induced asthma models following recombinant TL1A or anti-C-C motif chemokine receptor 8 (anti-CCR8) intervention. (J) Hematoxylin and eosin (HE) staining of lung tissue after allergen challenge and recombinant TL1A (rTL1A) or anti-CCR8 interventions. Black scale bar, 50 µm. (K) Total cell numbers in BAL samples. (L to N) Concentrations of CCL8, interleukin 4 (IL-4), and interleukin 13 (IL-13) in BAL samples from the different groups. Data are presented as mean ± SD from at least 2 independent experiments, with n = 5 to 6 mice per group per experiment. PCR, ELISA, and histological assessments were conducted by researchers blinded to treatment groups. Public dataset correlation (H) used n = samples from the GSE76262 dataset. * P < 0.05 and ** P < 0.01 compared with the respective groups.

Article Snippet: The recombinant proteins, small-molecular compounds, or inhibitors were purchased as indicated: recombinant mouse IL-13, IL-33, and TSLP protein (MCE); recombinant human IL-2, IL-4, TNF-α, and IFN-γ protein (Abbkine); poly(I:C) (Selleck); lipopolysaccharide (Sigma-Aldrich); IL-1β (Thermo Fisher); and small-molecule inhibitors BAY11-7082, LY294002, PD98059, SB203580, and SP600125 (Selleck).

Techniques: Expressing, Knockdown, Gene Expression, Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Knock-Out, Transgenic Assay, Recombinant, Staining