Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TSLP Interferes with Airway Tolerance by Suppressing the Generation of Antigen-specific Regulatory T cells 1

doi: 10.4049/jimmunol.1002503

Figure Lengend Snippet: A, TSLP suppressed murine iTreg differentiation in the presence of TGF-β1. Naïve CD4+CD62L+ T cells were isolated from spleens and lymph nodes, cultured with 10 μg/ml plate-bound anti-CD3 and 2 μg/ml anti-CD28 in the presence of 3 ng/ml TGF-β1 for 5 days. TSLP was used at 10 ng/ml. Data represent mean ± SEM (n = 3). B, TSLP suppressed human iTreg differentiation in the presence of TGF-β1. Naïve CD4+ T cells were isolated from PBMC and cultured in the same condition as (A). Human T cells showed considerable variability in responding to TSLP. A responder is shown on top, a non-responder on the bottom. The percentage inhibition of iTreg differentiation by TSLP from all 20 donor samples is indicated in the graph on the right. C, Purity of naïve CD4+ T cells isolated from human PBMC. Samples of PBMC and purified T cells from the same donor were stained with Blood Dendritic Cell Enumeration Kit (Miltenyi Biotec) and analyzed by flow cytometry. Data represent mean ± SEM (n = 6). D, Quantitative PCR analysis of the expression of human TSLP receptor TSLPR in activated CD4+ T cells, presented relative to naïve cells. Including of TSLP in culture (+TSLP) further enhanced TSLPR expression. Naïve CD4+ T cells were purified from PBMC and cultured in the same condition as (A). Data represent mean ± SEM (n = 6). *: p < 0.05; **: p < 0.01 One way analysis of variance.

Article Snippet: Since it is reported that human TSLP mainly exerts its effect through DCs and had little effect on CD4 + T cells ( 32 ), we examined DC contamination in our purified human naïve CD4+ T cells by Blood Dendritic Cell Enumeration Kit (Miltenyi Biotec).

Techniques: Isolation, Cell Culture, Inhibition, Purification, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing

Journal: Molecules

Article Title: Lignans from Machilus thunbergii as Thymic Stromal Lymphopoietin Inhibitors

doi: 10.3390/molecules26164804

Figure Lengend Snippet: The inhibitory effects of isolated compounds 1 – 3 on thymic stromal lymphopoietin (TSLP)/TSLP receptor (TSLPR) interaction by ELISA.

Article Snippet: For the pSTAT5 assay measuring TSLP inhibitory activity, recombinant human TSLP was purchased from R&D Systems (Minneapolis, MN, USA) and BD Cytofix/Cytoperm and Alexa Fluor 647 mouse anti-STAT5 (pY694) were purchased from BD Biosciences (San Diego, CA, USA).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Inhibition, Control

Journal: Molecules

Article Title: Lignans from Machilus thunbergii as Thymic Stromal Lymphopoietin Inhibitors

doi: 10.3390/molecules26164804

Figure Lengend Snippet: Molecular docking results of isolated compounds 1 – 3 on TSLP/TSLPR interaction.

Article Snippet: For the pSTAT5 assay measuring TSLP inhibitory activity, recombinant human TSLP was purchased from R&D Systems (Minneapolis, MN, USA) and BD Cytofix/Cytoperm and Alexa Fluor 647 mouse anti-STAT5 (pY694) were purchased from BD Biosciences (San Diego, CA, USA).

Techniques: Isolation

Journal: Molecules

Article Title: Lignans from Machilus thunbergii as Thymic Stromal Lymphopoietin Inhibitors

doi: 10.3390/molecules26164804

Figure Lengend Snippet: Molecular docking results of isolated compounds 1 – 3 against human TSLP in complex with TSLPR and interleukin-7 receptor α chain (IL-7Rα): ( A ) three-dimensional (3D) docking image of compound 1 ; ( B ) two-dimensional (2D) docking image of compound 1 ; ( C ) 3D docking image of compound 2 ; ( D ) 2D image picture of compound 2 ; ( E ) 3D docking image of compound 3 ; ( F ) 2D docking picture of compound 3 .

Article Snippet: For the pSTAT5 assay measuring TSLP inhibitory activity, recombinant human TSLP was purchased from R&D Systems (Minneapolis, MN, USA) and BD Cytofix/Cytoperm and Alexa Fluor 647 mouse anti-STAT5 (pY694) were purchased from BD Biosciences (San Diego, CA, USA).

Techniques: Isolation

Journal: PLoS ONE

Article Title: Loss of Grainy Head-Like 1 Is Associated with Disruption of the Epidermal Barrier and Squamous Cell Carcinoma of the Skin

doi: 10.1371/journal.pone.0089247

Figure Lengend Snippet: (A) Blood levels of TSLP in Grhl1 +/+ (gray bar) and Grhl1 −/− mice (blue bar), measured with ELISA kit. (B) Levels of expression of antimicrobial peptides S100A8 and S100A9 in the epidermis of Grhl1 +/+ (gray bar) and Grhl1 −/− mice (blue bar), measured with Q-RT-PCR. (C) Representative skin sections of Grhl1 +/+ (top panel) and Grhl1 −/− mice (bottom panel) stained with toluidine blue to visualize dermal mast cells (purple cells). Scale bars represent 200 µm. (D) Quantification of skin infiltration with mast cells for Grhl1 +/+ (gray bar) and Grhl1 −/− mice (blue bar), estimated as numbers of stained cells per 1 mm 2 area of 10 µm thick skin section (using ImageJ software). (A, B, D) Significance (Student’s t-test, p-value) is shown above bars.

Article Snippet: The blood level of TSLP was measured using enzyme-linked immunosorbent assay (ELISA) kit: Quantikine Mouse TSLP (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions, on groups of seven Grhl1 −/− and five Grhl1 +/+ animals, at the age of about 4 months.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Software

Journal: Biomedical Reports

Article Title: Therapeutic potential of a systemically applied humanized monoclonal antibody targeting Toll‑like receptor 2 in atopic‑dermatitis‑like skin lesions in a mouse model

doi: 10.3892/br.2024.1919

Figure Lengend Snippet: Figure 2. Effect of systemic administration of Toma on DNCB‑induced FLG and TSLP expression in BALB/c mice. (A and B) Paraffin‑embedded tissue sections prepared on day 20 were subjected to immunofluorescence staining with (A) anti‑FLG and (B) anti‑TSLP antibodies with a rhodamine‑red‑X‑conjugated secondary antibody (red). The nuclei were counterstained with Hoechst 33258 (blue). Scale bars, 100 µm. ImageJ software was used to measure fluorescent signals (right graphs). The data are expressed as the mean ± SD. (n=6). *P<0.05 and **P<0.01 by Tukey's multiple comparisons test. Toma; Tomaralimab; DNCB, 2,4‑dinitrochlorobenzene; FLG, filaggrin; TSLP, thymic stromal lymphopoietin; NS, not significant.

Article Snippet: Anti‐thymic stromal lymphopoietin (TSLP) Ab (cat. no. NBP1‐76754) was obtained from Novus Biologicals, LLC and anti‐HMGB1 Ab (cat.

Techniques: Expressing, Immunofluorescence, Staining, Software

Journal: Biomedical Reports

Article Title: Therapeutic potential of a systemically applied humanized monoclonal antibody targeting Toll‑like receptor 2 in atopic‑dermatitis‑like skin lesions in a mouse model

doi: 10.3892/br.2024.1919

Figure Lengend Snippet: Figure 4. Effect of DNCB on the expression of pro‑inflammatory cytokines in HaCaT keratinocytes. (A and B) The effect of DNCB on the expression of pro‑inflammatory cytokines was examined using immunoblot analysis based on different (A) doses and (B) time‑points. The band intensities were measured using ImageJ software. The data are expressed as the mean ± SD (n=3). *P<0.05 and **P<0.01 by Dunnett's multiple comparisons test. DNCB, 2,4‑dinitrochlo‑ robenzene; TSLP, thymic stromal lymphopoietin; IL, interleukin; NS, not significant.

Article Snippet: Anti‐thymic stromal lymphopoietin (TSLP) Ab (cat. no. NBP1‐76754) was obtained from Novus Biologicals, LLC and anti‐HMGB1 Ab (cat.

Techniques: Expressing, Western Blot, Software

Journal: Biomedical Reports

Article Title: Therapeutic potential of a systemically applied humanized monoclonal antibody targeting Toll‑like receptor 2 in atopic‑dermatitis‑like skin lesions in a mouse model

doi: 10.3892/br.2024.1919

Figure Lengend Snippet: Figure 5. Effect of Toma on the inhibition of DNCB‑induced pro‑inflammatory cytokines in HaCaT keratinocytes. (A and B) The effect of Tomaralimab on the inhibition of DNCB‑induced pro‑inflammatory cytokines was examined using (A) reverse transcription‑quantitative PCR and (B) immunoblot analysis. The band intensities were measured using ImageJ software. The data are expressed as the mean ± SD (n=3). *P<0.05 and **P<0.01 by Dunnett's multiple comparisons test. Toma, Tomaralimab; DNCB, 2,4‑dinitrochlorobenzene; TSLP, thymic stromal lymphopoietin; IL, interleukin; NS, not significant.

Article Snippet: Anti‐thymic stromal lymphopoietin (TSLP) Ab (cat. no. NBP1‐76754) was obtained from Novus Biologicals, LLC and anti‐HMGB1 Ab (cat.

Techniques: Inhibition, Western Blot, Software

Journal: Arthritis and rheumatism

Article Title: A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

doi: 10.1002/art.23965

Figure Lengend Snippet: Figure 1. Induction of thymic stromal lymphopoietin (TSLP) mRNA and protein expression in mouse intervertebral disc cells via the NF-B pathway. A, Left, Mouse intervertebral discs were cultured in the presence or absence of 10 ng/ml tumor necrosis factor (TNF), interleukin-1 (IL-1), and 1 g/ml lipopolysaccharide (LPS) for the indicated periods of time. RNA was then extracted, and real-time reverse transcription– polymerase chain reaction was performed using specific primers for TSLP and GAPDH. The ratio for expression of each gene to that of GAPDH was calculated, and the relative expression levels are shown. Right, Mouse intervertebral discs were cultured in the presence or absence of 10 ng/ml TNF, IL-1, IL-6, and transforming growth factor (TGF), and 1 g/ml LPS for 72 hours. The culture supernatants were collected, and TSLP concentrations were measured by enzyme-linked immunosorbent assay (ELISA). B, For immunohistochemical examination of mouse intervertebral disc tissue, tissue sections were stained with anti-mouse TSLP antibody as described in Materials and Methods. No positive immunohistologic cell staining following stimulation with TNF was seen in control IgG-treated sections (goat-Ab). C, Mouse intervertebral discs obtained from wild-type (WT), TNF receptor I (TNFRI)–deficient (RI-knockout [RIKO]), and TNFRII-deficient (RIIKO) mice were cultured in the presence or absence of 10 ng/ml TNF or 1 g/ml LPS for 72 hours. Culture supernatants were collected, and TSLP concentrations were measured by ELISA. D, Mouse intervertebral discs obtained from wild-type mice were cultured in the presence or absence of 10 ng/ml TNF, IL-1, or 1 g/ml LPS with or without 1 M IMD-0354 or 10 M MG132 for 72 hours. Culture supernatants were collected, and TSLP concentrations were measured by ELISA. Values in A, C, and D are the mean SD results and are representative of 3 independent experiments. P 0.05 versus control. NS no stimulation.

Article Snippet: Recombinant mouse tumor necrosis factor (TNF ), mouse IL-1 , mouse IL-6, human transforming growth factor (TGF ), mouse TSLP, mouse or human TSLPR-Fc chimera, anti-mouse and anti-human TSLP neutralizing antibody and anti-mouse MCP-1 antibody, and control goat IgG were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Cell Culture, Reverse Transcription, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Control, Knock-Out

Journal: Arthritis and rheumatism

Article Title: A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

doi: 10.1002/art.23965

Figure Lengend Snippet: Figure 2. TSLP-induced expression of monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 (MIP-1), and macrophage colony-stimulating factor (M-CSF) in mouse intervertebral disc cells. A, Mouse intervertebral disc culture lysates were subjected to Western blot analysis with antibodies specific for TSLPR and -actin. Mouse TSLPR-Fc chimera protein (mTSLP-R) was used as a positive control, and human TSLPR-Fc chimera protein (hTSLP-R) was used as a negative control. B, Mouse intervertebral discs were cultured in the presence or absence of 10 ng/ml TSLP for 72 hours. The supernatants were collected and subjected to a cytokine protein array. The table shows the corresponding cytokines on the protein array membrane. C, Mouse intervertebral discs were cultured in the presence or absence of 10 ng/ml TSLP for 72 hours. Supernatants were collected, and MCP-1, MIP-1, M-CSF, and RANTES concentrations were measured by ELISA. Values are the mean and SD. D, For immunohistochemical examination of mouse intervertebral disc tissue, tissue sections were stained with anti-mouse MCP-1 antibody, as described in Materials and Methods. No positive immunohistologic cell staining following stimulation with TNF was seen in control IgG-treated sections (goat-Ab). Results are representative of 3 independent experiments. P 0.05 versus control. GM-CSF granulocyte–macrophage CSF; SDF-1 stromal cell–derived factor 1; IFN- interferon-; TCA-3 T cell–attracting chemokine 3; I-TAC interferon-inducible T cell chemoattractant; TECK thymus-expressed chemokine; KC cytokine-induced neutrophil chemoattractant; TIMP-1 tissue inhibitor of metalloproteinases 1; LIX lipopolysaccharide-induced CXC chemokine; BLC B lymphocyte chemoattractant; sTNF R1 soluble TNF receptor I; GCSF granulocyte CSF (see Figure 1 for other definitions).

Article Snippet: Recombinant mouse tumor necrosis factor (TNF ), mouse IL-1 , mouse IL-6, human transforming growth factor (TGF ), mouse TSLP, mouse or human TSLPR-Fc chimera, anti-mouse and anti-human TSLP neutralizing antibody and anti-mouse MCP-1 antibody, and control goat IgG were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Positive Control, Negative Control, Cell Culture, Protein Array, Membrane, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Control, Derivative Assay

Journal: Arthritis and rheumatism

Article Title: A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

doi: 10.1002/art.23965

Figure Lengend Snippet: Figure 3. TSLP induction of MCP-1 production in mouse intervertebral disc cells primarily via the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. A, Intervertebral discs obtained from wild-type mice were cultured in the presence or absence of 10 ng/ml TSLP and/or 10 M SP600125 (JNK inhibitor), 1 M SB203580 (p38 inhibitor), and 10 M LY294002 (PI 3-kinase inhibitor) for 72 hours. Culture supernatants were collected, and the TSLP concentrations were measured by ELISA. B, Intervertebral discs obtained from wild-type mice were cultured in the presence or absence of 10 ng/ml TSLP with or without 10 M LY294002 (PI 3-kinase inhibitor) for the indicated periods of time. Culture supernatants were collected, and TSLP concentrations were measured by ELISA. C, Intervertebral discs obtained from wild-type mice were cultured in the presence of 10 M LY294002 for 72 hours. After washing and replacing the culture medium without LY294002, the discs were then cultured in the presence or absence of 10 ng/ml TSLP for 72 hours. Culture supernatants were collected, and TSLP concentrations were measured by ELISA. D, Mouse intervertebral disc cells were stimulated with 10 ng/ml TSLP for the indicated periods of time (top) and were also stimulated with 10 ng/ml TSLP in the presence or absence of the indicated doses of LY294002 for 60 minutes (bottom). Culture lysates were subjected to Western blot analysis with antibodies specific for phosphorylated Akt. The same membrane was used for Western blot analysis with nonphosphorylated Akt antibody after stripping of the phosphorylated Akt antibody (top and bottom). Results in A–C are the mean and SD and are representative of 3 independent experiments. P 0.05. See Figure 1 for other definitions.

Article Snippet: Recombinant mouse tumor necrosis factor (TNF ), mouse IL-1 , mouse IL-6, human transforming growth factor (TGF ), mouse TSLP, mouse or human TSLPR-Fc chimera, anti-mouse and anti-human TSLP neutralizing antibody and anti-mouse MCP-1 antibody, and control goat IgG were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Membrane, Stripping Membranes

Journal: Arthritis and rheumatism

Article Title: A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

doi: 10.1002/art.23965

Figure Lengend Snippet: Figure 4. Induction of macrophage migration via monocyte chemotactic protein 1 (MCP-1) by TSLP-activated intervertebral disc culture supernatants. Macrophages (1.5 106) in the upper chamber of a tissue culture insert were allowed to migrate toward the lower chamber, where 3 discs per 96-well plate had been cultured in 1 ml of Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum in the presence or absence of 10 ng/ml TNF or TSLP with or without 10 g/ml anti–MCP-1 antibody for 24 hours. After the incubation of macrophages and TSLP- or TNF- activated disc cultures for 6 hours at 37°C, the membrane was removed, and nonmigrated cells on the upper surface of the membrane were removed. Cells were fixed and stained with Giemsa solution. Cells on the lower surface of the membrane were counted in 4 high-power fields. A, Representative photomicrographs of migrated macrophages on the lower surface of the membrane. B, Quantitative analysis of the number of macrophages that migrated to the lower surface of the membrane. Results are the mean and SD and are representative of 3 independent experiments. P 0.05. See Figure 1 for other definitions.

Article Snippet: Recombinant mouse tumor necrosis factor (TNF ), mouse IL-1 , mouse IL-6, human transforming growth factor (TGF ), mouse TSLP, mouse or human TSLPR-Fc chimera, anti-mouse and anti-human TSLP neutralizing antibody and anti-mouse MCP-1 antibody, and control goat IgG were purchased from R&D Systems (Minneapolis, MN).

Techniques: Migration, Cell Culture, Modification, Incubation, Membrane, Staining

Journal: Arthritis and rheumatism

Article Title: A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

doi: 10.1002/art.23965

Figure Lengend Snippet: Figure 5. Partial mediation of TNF-induced monocyte chemotactic protein 1 (MCP-1) production in mouse intervertebral disc cells by TSLP. Mouse intervertebral discs were cultured in the presence or absence of 10 ng/ml TNF for 72 hours, with or without 10 g/ml mouse or human TSLPR-Fc chimera (mTSLP-R/FC and hTSLP-R/Fc, respectively), or 10 g/ml anti-mouse TSLP antibody or anti-human TSLP antibody. Culture supernatants were collected, and MCP-1 concentrations were measured by ELISA. Results are the mean and SD and are representative of 3 independent experiments. P 0.05. See Figure 1 for other definitions.

Article Snippet: Recombinant mouse tumor necrosis factor (TNF ), mouse IL-1 , mouse IL-6, human transforming growth factor (TGF ), mouse TSLP, mouse or human TSLPR-Fc chimera, anti-mouse and anti-human TSLP neutralizing antibody and anti-mouse MCP-1 antibody, and control goat IgG were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

doi: 10.1002/art.23965

Figure Lengend Snippet: Figure 6. Thymic stromal lymphopoietin (TSLP) and monocyte chemotactic protein 1 (MCP-1) expression in herniated disc tissue. Ten herniated disc tissue specimens obtained from 10 patients who underwent surgical resections for a herniated disc were immunohistochemically stained with anti-TSLP and anti–MCP-1 antibodies. No positive immunohistologic cell staining was seen in control IgG-treated sections. A, Representative photomicrographs from one herniated disc specimen. Positive staining is indicated as a brown color. B, Quantitative analysis of the number of hematoxylin-staining cells and TSLP- or MCP-1–immunoreactive cells in 4 high-power fields (100) of herniated disc tissue (n 10). Values are the mean SD. Positive immunoreactive; negative no staining.

Article Snippet: Recombinant mouse tumor necrosis factor (TNF ), mouse IL-1 , mouse IL-6, human transforming growth factor (TGF ), mouse TSLP, mouse or human TSLPR-Fc chimera, anti-mouse and anti-human TSLP neutralizing antibody and anti-mouse MCP-1 antibody, and control goat IgG were purchased from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Staining, Control

Journal: Annals of the rheumatic diseases

Article Title: A profibrotic role for thymic stromal lymphopoietin in systemic sclerosis

doi: 10.1136/annrheumdis-2012-202279

Figure Lengend Snippet: Expression of thymic stromal lymphopoietin (TSLP) in human systemic sclerosis (SSc) skin and bleomycin-induced skin fibrosis. Immunohistochemical analysis of TSLP expression in human SSc and healthy skin sections. (A) Thymus (positive control); (B) healthy skin; (C) SSc skin; (D) SSc epidermis; (E) SSc skin immunostained with non-immune serum control. Insets in C and D show fibroblast and mast cell immunostaining with higher magnification. TSLP expression in bleomycin-treated mice: (F) control (saline) skin immunostained with non-immune serum control; (G) control (saline) skin; (H) bleomycin-injected mouse skin; (I) quantitative RT-PCR analysis of TSLP mRNA expression in bleomycin-injected skin (Bleo) compared with control (saline injected) skin (*p<0.001). Data are representative of three independent experiments with 10 mice per group. Bar 100 μm (A–C), 50 μm (D–H).

Article Snippet: Additional IHC blocking controls were performed by preincubating anti-TSLP antibody with TSLP immunising peptide (Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemical staining, Positive Control, Control, Immunostaining, Saline, Injection, Quantitative RT-PCR