Journal: Frontiers in Immunology

Article Title: Thymic stromal lymphopoietin promotes abdominal aortic aneurysm formation by regulating macrophage polarization

doi: 10.3389/fimmu.2026.1767913

Figure Lengend Snippet: Therapeutic neutralization of TSLP protects against AAA development. (A) Schematic diagram of the experimental protocol for anti-mouse TSLP antibody (anti-TSLP) administration in the PPE-induced AAA model. (B) Comparison of the maximum abdominal aortic diameter in normal saline-treated and TSLP-treated mice at day 14 (n=6 mice per group). (C, D) Representative histological staining and corresponding quantification for Elastica Van Gieson (EVG) (n=6 mice per group). (E–J) Representative images and quantitative analysis of immunohistochemical staining for CD68 (E, F) , MMP2 (G, H) and MMP9 (I, J) expression in the aortic wall. Positive areas are presented as a percentage of the total area (n=6 mice per group). Scale bars: (A) 1mm; (C) 500 μm; (E, G, I) 200 μm (top), 50 μm (bottom). All quantitative data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired two-tailed Student’s t-test). Ctrl, control. Red arrows indicate representative positive staining areas.

Article Snippet: Anti-TSLP antibody treatment: Anti-TSLP antibody (HY-P990150, MCE, USA) was administered via intraperitoneal injection starting on day 1 after PPE induction at a dose of 20 mg/kg, and then every other day thereafter.

Techniques: Neutralization, Comparison, Saline, Staining, Immunohistochemical staining, Expressing, Two Tailed Test, Control

Journal: bioRxiv

Article Title: Distinct roles for thymic stromal lymphopoietin (TSLP) and IL-33 in experimental eosinophilic esophagitis

doi: 10.1101/2025.02.25.640192

Figure Lengend Snippet: Experimental EoE was induced in wild type (WT) mice using oxazolone (OXA). Starting on day 19, the mice received two intraperitoneal injections per week of isotype control antibodies or anti-TSLP (clones M702 or clone 28F12, A). On day 36, the mice were euthanized, and esophageal tissues were fixed, paraffin embedded, and slides were generated. The slides were stained with anti-major basic protein (MBP, B) and esophageal eosinophils were quantified (C). Epithelial cell proliferation was determined using anti-Ki67 staining (D, E). H&E-stained slides (G) were analysed for lamina propria (F) as well as epithelial (H) thickness. Esophageal vascularization was determined using anti-CD31 staining (I, J). Single cell suspensions of esophageal tissue were obtained from oxazolone-challenged isotype control or anti-TSLP (M702)-treated mice using enzymatic digestion and the levels of eosinophils and CD4 + T cells determined by flow cytometry (K, L). Representative photomicrographs of anti-MBP (B), anti-Ki-67 (D), H&E (G) and anti-CD31 (I) are shown. Data are presented as mean ± SEM and are representative of n=3 experiments conducted with 10-12 mice per group, ns-nonsignificant, *-p<0.05, **-p<0.01, ***-p<0.001, ****-p<0.0001 .

Article Snippet: To substantiate these findings, TSLP was neutralized using an additional clone of anti-TSLP (BioXcell, clone 28F12).

Techniques: Control, Clone Assay, Generated, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Distinct roles for thymic stromal lymphopoietin (TSLP) and IL-33 in experimental eosinophilic esophagitis

doi: 10.1101/2025.02.25.640192

Figure Lengend Snippet: Experimental EoE was induced in wild type (WT) mice. Starting on day 19, the mice received two intraperitoneal injections per week of isotype control antibodies or anti-TSLP (clones M702 or clone 28F12). On day 36, the mice were euthanized, and esophageal tissue was obtained and subjected to bulk RNA sequencing. PCA plot of vehicle- and oxazolone (OXA)-challenged mice treated with either isotype control or anti-TSLP antibodies (clones M702 and/or 28F12) is shown (A). Heat plot analysis of all differentially expressed genes (absolute fold change > 2, p<0.05) (B) is presented. Analysis of the top differentially expressed secreted factors, cytokine receptors, adhesion molecules and extracellular matrix components is shown (C-D). Gene Set Enrichment Analysis (GSEA) and STRING analysis were performed on the DEGs, and comparison of OXA-challenged vehicle-treated mice vs. OXA-challenged anti-TSLP treated mice was performed (E-H). The top up and down regulated pathways, which were regulated by TSLP are presented (E-F). In C, D and E, each lane represents a different mouse.

Article Snippet: To substantiate these findings, TSLP was neutralized using an additional clone of anti-TSLP (BioXcell, clone 28F12).

Techniques: Control, Clone Assay, RNA Sequencing, Comparison

Journal: bioRxiv

Article Title: Telocytes link epithelial nutrient sensing with amplification of the ILC2-tuft cell circuit

doi: 10.1101/2024.10.14.618111

Figure Lengend Snippet: (A) Gene-targeting strategy for the flox and reporter of Tslp (Flare-TSLP) mouse. Frt, target site for FLIPASE recombinase; IRES, internal ribosomal entry site; loxP , target site for Cre recombinase; pA, bovine growth hormone poly(A) tail; tdRFP, tandem-dimer red fluorescent protein, or tdTomato; UTR, untranslated region. (B) Representative imaging of jejunum ( top ) and ileum ( bottom ) of Flare-TSLP mice or non-reporter control mice. Red, TSLP-tdTomato; Green, PDGFRɑ; White, EPCAM; Blue, DAPI. 20x objective. Arrowheads indicate TSLP-tdTomato + cells. Scale bar, 50 µm. ( C )( D ) Flow cytometric analysis of endothelial and other stromal populations in jejunum and ileum small intestine lamina propria (siLP) in Flare-TSLP mice. ( C ) Percentage of total TSLP-tdTomato + in CD45 - population. ( D ) Percentage of endothelial cells and other stromal within TSLP-tdTomato + cells. Cells were stained with anti-CD31 and anti-Podoplanin (PDPN) antibodies. Lymphatic endothelial cells are CD45 - PDPN + CD31 + and fibroblasts are CD45 - PDPN + CD31 - . (E) RT-qPCR analysis of purified CD45 - EPCAM + cells, CD45 - EPCAM + SiglecF + tuft cells, CD45 - CD31 - PDPN + total stromal cells, CD45 - CD31 - PDPN + TSLP-tdTomato + cells, CD45 - CD31 + PDPN + lymphatic endothelial and CD45 + PDPN - hematopoietic cells from small intestinal epithelial fraction or siLP (whole tissue) of Flare-TSLP mice. Gene expression normalized to 18S . (F) Representative imaging of jejunum ( middle ) and ileum ( bottom ) of Flare-TSLP; Lgr5 -eGFP dual reporter mice or Flare-TSLP single reporter (Ctrl) mice ( top ). Red, TSLP-tdTomato; Green, Lgr5 -eGFP; White, EPCAM; Blue, DAPI. 20x objective. Scale bar, 50 µm. (G) Representative imaging of CD201 and CD34 expression in jejunum ( top ) and ileum ( bottom ) of Flare-TSLP mice. Red, TSLP-tdTomato; Green, CD201 ( left ) or CD34 ( right ); Blue, DAPI. 20x objective. Scale bar, 50 µm. (H) RT-qPCR analysis of purified TSLP-tdTomato + CD201 + CD31 - (telocytes), and TSLP-tdTomato + CD34 + CD31 - (trophocytes), CD201 + CD31 - , and CD34 + CD31 - stromal cells from siLP (whole tissue) of Flare-TSLP mice. Gene expression normalized to 18S . All data are biological replicates, n≥3. Data are representative of at least two independent experiments. Error bars indicate samples mean± SEM.

Article Snippet: Mouse TSLP antibody for flow cytometric analysis was labeled using Alexa FluorTM 488 Antibody Labeling Kit (ThermoFisher, Cat# A20181) following the manufacture protocol.

Techniques: Imaging, Control, Staining, Quantitative RT-PCR, Purification, Gene Expression, Expressing

Journal: bioRxiv

Article Title: Telocytes link epithelial nutrient sensing with amplification of the ILC2-tuft cell circuit

doi: 10.1101/2024.10.14.618111

Figure Lengend Snippet: ( A ) Representative imaging showing jejunum ( top ) and ileum ( bottom ) of Flare-TSLP mice or non-reporter control mice. Red, TSLP-tdTomato; Green, PDPN; Blue, DAPI. 20x objective. Scale bar, 50 µm. Right panel , max intensity projection of three - dimensional imaging showing jejunual tissues from Flare-TSLP mice ; scale bar, 100 µm. ( B )-( D ) Flow cytometric analysis of CD45 + , endothelial and non-endothelial stromal populations in jejunal and ileal lamina propria in Flare-TSLP or non-reporter control (ctrl) mice. ( B ) CD45 + cells were CD31 + specialized endothelial cells. CD45 - CD31 - stromal cells were PDGFRɑ + cKit - (non-Cajal cells, which are cKit + ). ( C ) Proportions of CD45 + CD31 + , CD45 + CD31 - , CD45 - CD31 + , and CD45 - CD31 - cells in TSLP-tdTomato + cells represented by pie charts. ( D ) TSLP-tdTomato + CD45 - CD31 + cells were PDPN + consistent with lymphatic endothelial cells. TSLP-tdTomato + CD45 + cells are CD31 + FSC hi large cells consistent with a non-hematopoietic lineage. ( E ) Representative imaging showing back skin of Flare-TSLP mice treated with ethanol or MC903 for 7 days. Red, TSLP-tdTomato; Blue, DAPI. 20x objective. Scale bar, 100 µm. ( F ) Flow cytometric analysis of epithelial TSLP expression in proximal small intestine in naive Flare-TSLP or non-reporter mice or on day 7 and 12 post N. brasiliensis infection. d.p.i., day post infection. ( G ) Representative imaging of jejunum in Flare-TSLP or non-reporter mice on day 5 after N. brasiliensis infection. Red, TSLP-tdTomato; Green, PDGFRɑ; White, EPCAM; Blue, DAPI. 20x objective. Scale bar, 100 µm. All data are biological replicates, n≥3. Data are representative of at least two independent experiments.

Article Snippet: Mouse TSLP antibody for flow cytometric analysis was labeled using Alexa FluorTM 488 Antibody Labeling Kit (ThermoFisher, Cat# A20181) following the manufacture protocol.

Techniques: Imaging, Control, Expressing, Infection

Journal: bioRxiv

Article Title: Telocytes link epithelial nutrient sensing with amplification of the ILC2-tuft cell circuit

doi: 10.1101/2024.10.14.618111

Figure Lengend Snippet: ( A ) ( B ) CD201 and CD34 staining of TSLP-tdTomato + cells in jejunal and ileal lamina propria (LP) of Flare-TSLP mice. ( A ) Representative flow cytometric analysis. ( B ) Quantification of percentage of CD34 + and CD34 - CD201 + in TSLP-tdTomato + populations. ( C ) Representative imaging of CD201 and CD34 expression in proximal and distal small intestine of Flare-TSLP; Lgr5 -eGFP dual reporter mice. Scale bar, 50 µm. ( D ) Gating strategy for sorting of TSLP-tdTomato + CD201 + CD31 - , and TSLP-tdTomato + CD34 + CD31 - cells from small intestinal LP (siLP, whole tissue) of Flare-TSLP mice. ( E ) RT-qPCR analysis of purified TSLP-tdTomato + CD201 + CD31 - (telocytes), and TSLP-tdTomato + CD34 + CD31 - (trophocytes), CD201 + CD31 - , and CD34 + CD31 - stromal cells from siLP (whole tissue) of Flare-TSLP mice. All data are biological replicates, n≥3. Data are representative of at least two independent experiments. Error bars indicate samples mean± SEM.

Article Snippet: Mouse TSLP antibody for flow cytometric analysis was labeled using Alexa FluorTM 488 Antibody Labeling Kit (ThermoFisher, Cat# A20181) following the manufacture protocol.

Techniques: Staining, Imaging, Expressing, Quantitative RT-PCR, Purification

Journal: bioRxiv

Article Title: Telocytes link epithelial nutrient sensing with amplification of the ILC2-tuft cell circuit

doi: 10.1101/2024.10.14.618111

Figure Lengend Snippet: (A) Schematic of the protocol for measuring tissue TSLP after feeding. Mice were fasted for 16 hr overnight (O/N) before oral gavage with 500 μl food slurry (refed) or water as volumetric control (sham). Tissues were harvested as indicated. (B) Percentage of TSLP-tdTomato + cells among CD45 - cells in proximal jejunal small intestine lamina propria (siLP) by flow cytometric analysis after oral gavage at 2 hr. (C) ELISA of TSLP protein recovered from proximal jejunal tissue explants after oral gavage at 2 hr in Pou2f3 -/- or WT mice. (D) Schematic of the protocol for measuring siLP ILC2 activation after feeding. Mice were fasted 16 hr overnight and given access to standard chow and water ad libitum (refed) or maintained on water control (fasted). Tissues were harvested 4 hr later. ( E )( F ) Percentage of IL-13 (Smart13) + ILC2 among ILC2s in proximal jejunal LP in Tslpr +/+ Red5Smart13 or Tslpr -/- Red5Smart13 mice after 16 hr fasting followed by 4 hr refeeding ad libitum . ( E ) Representative flow plots. ( F ) Quantitation across experiments. ILC2s were gated as Lin - CD45 + IL-5 (Red5) + cells. ( G )( H ) Percentage of IL-13 (Smart13) + ILC2 among ILC2s in proximal jejunal LP 4 hr after refeeding ad libitum in overnight fasted Tslp fl/fl Pdgfrɑ CreERT2+ Smart13 or littermate control Pdgfrɑ +/+ Smart13 mice (ctrl) post-tamoxifen. ( G ) Representative flow plots. ( H ) Quantitation across experiments. ILC2s were gated on Lin - CD45 + GATA3 + cells. (I) Percentage of IL-13 (Smart13) + ILC2 among ILC2s in proximal jejunal LP in Tslpr fl/fl Il5 Cre+ (Red5)Smart13 mice after 16 hr fasting followed by 4 hr refeeding ad libitum or maintained on water control. ILC2s were gated as Lin - CD45 + IL-5 (Red5) + cells. All data are biological replicates, n≥3. Data are representative of at least two independent experiments. Error bars indicate samples mean± SEM.*P<0.03, **P<0.002, ***P<0.0002.

Article Snippet: Mouse TSLP antibody for flow cytometric analysis was labeled using Alexa FluorTM 488 Antibody Labeling Kit (ThermoFisher, Cat# A20181) following the manufacture protocol.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Activation Assay, Quantitation Assay

Journal: bioRxiv

Article Title: Telocytes link epithelial nutrient sensing with amplification of the ILC2-tuft cell circuit

doi: 10.1101/2024.10.14.618111

Figure Lengend Snippet: ( A ) Percentage of TSLP-tdTomato + cells among CD45 - cells in ileal lamina propria by flow cytometric analysis in fasted mice at 2 hr after oral gavage of food (refed) or water as volumetric control (sham). ( B ) ELISA of TSLP protein recovered from proximal jejunal ( left ) and distal ileal ( right ) tissue homogenates after oral gavage of food at designated times in fasted wild type (WT) mice. (B) ELISA of TSLP protein recovered from jejunum and distal ileal tissue homogenates from fasted Pou2f3 -/- mice at 2 hr after oral food gavage. (D)(E) Single-cell analysis of a human intestinal dataset ( 24 ). ( D ) UMAP representing cell clustering. ( E ) Gene expression levels of TSLP , GLP2R , FOXL1 , GREM1 , CD34 , PROCR , BMP5 , RSPO3 , WNT2B , WNT5B , PDGFRA , and PDPN . ( F ) Representative imaging showing a preproglucagon + EEC (L cell) and TSLP-tdTomato + telocyte in proximity in the jejunum of Gcg -YFP; Flare-TSLP dual reporter mice after 16 hr overnight fasting followed by refeeding ad libitum for 4 hr. Red, TSLP-tdTomato; Green, Gcg -YFP; White, EPCAM; Blue, DAPI. 20x objective. Scale bar, 50 µm. All data are biological replicates, n≥3. Data are representative of at least two independent experiments. Error bars indicate samples mean± SEM. **P<0.002.

Article Snippet: Mouse TSLP antibody for flow cytometric analysis was labeled using Alexa FluorTM 488 Antibody Labeling Kit (ThermoFisher, Cat# A20181) following the manufacture protocol.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Single-cell Analysis, Gene Expression, Imaging

Journal: bioRxiv

Article Title: Telocytes link epithelial nutrient sensing with amplification of the ILC2-tuft cell circuit

doi: 10.1101/2024.10.14.618111

Figure Lengend Snippet: ( A ) Heatmaps representing expression levels of gut hormone receptors in telocytes , trophocytes , Foxl1 + telocytes and Foxl1 - stromal cells from available mouse datasets. ( B )( C ) Single-cell analysis of an available human intestinal dataset . ( B ) UMAP representing cell clustering. ( C ) TSLP ( left ) and GLP2R ( right ) expression levels. ( D ) RT-qPCR analysis of Glp1r ( left ) and Glp2r ( right ) expression on purified gut EPCAM + epithelial or TSLP-tdTomato + stromal cells. ( E ) Percentage of TSLP-tdTomato + CD45 - cells in proximal jejunal lamina propria (LP) 2 hr after GLP-2[Gly2] injections as assessed by flow cytometric analysis. Veh, vehicle ( F ) ELISA for TSLP protein recovered from proximal jejunal tissue explants in Glp2r -/- or wildtype (WT) mice 2 hr after GLP-2[Gly2] or vehicle control injections. ( G ) Flow cytometric analysis of percentage of total TSLP-tdTomato + CD45 - cells in proximal jejunal LP from fasted Glp2r -/- Flare-TSLP mice after oral food gavage at 2 hr. ( H ) ELISA for recovered TSLP protein in proximal jejunal homogenates from fasted Glp2r -/- mice after oral gavage at 2 hr. ( I ) Quantification of percentage of IL-13 (Smart13) + ILC2 among proximal jejunal LP ILC2s from fasted Glp2r -/- Smart13 mice after 16 hr fasting followed by 4 hr refeeding ad libitum or maintained on water control. ILC2s were gated on Lin - CD45 + GATA3 + cells. ( J ) Quantification of percentage of IL-13 (Smart13) + ILC2s among proximal jejunal LP ILC2s in Tslpr +/+ Red5Smart13 or Tslpr -/- Red5Smart13 mice after 3 daily subcutaneous (s.c.) injections of GLP-2[Gly2]. ILC2s were gated on Lin - CD45 + IL-5(Red5) + cells. ( K ) Quantification of percentage of IL-13 (Smart13) + ILC2s among proximal jejunal LP ILC2s in Tslpr fl/fl Il5 Cre+ Smart13 after 3 daily subcutaneous (s.c.) injections of GLP-2[Gly2]. ILC2s were gated on Lin - CD45 + IL-5(Red5) + cells. ( L ) ( M ) Flow cytometric analysis of percentage of IL-13 (Smart13) + ILC2 among proximal jejunal LP ILC2s in Tslp fl/fl Pdgfrɑ CreERT2+ Smart13 or littermate Pdgfrɑ +/+ Smart13 controls (ctrl) post-tamoxifen followed by 3 daily s.c. injections of GLP-2[Gly2]. ( L ) Representative flow plots. ( M ) Quantitation. ILC2s were gated as Lin - CD45 + GATA3 + cells. All data are biological replicates, n≥3. Error bars indicate samples mean± SEM. *P<0.03, ****P<0.00001.

Article Snippet: Mouse TSLP antibody for flow cytometric analysis was labeled using Alexa FluorTM 488 Antibody Labeling Kit (ThermoFisher, Cat# A20181) following the manufacture protocol.

Techniques: Expressing, Single-cell Analysis, Quantitative RT-PCR, Purification, Enzyme-linked Immunosorbent Assay, Control, Quantitation Assay

Journal: bioRxiv

Article Title: Telocytes link epithelial nutrient sensing with amplification of the ILC2-tuft cell circuit

doi: 10.1101/2024.10.14.618111

Figure Lengend Snippet: ( A )-( D ) Flow cytometric analysis of TSLP-tdTomato + cells in jejunal and ileal lamina propria (LP) at 2 hr after GLP-2[Gly2] injections. ( A ) Percentage of total TSLP-tdTomato + cells among CD45 - cells in ileal LP. Veh, vehicle. ( B ) Proportion of endothelial and other stromal populations within TSLP-tdTomato + cells in jejunum ( left ) and in ileum ( right ). (C)(D ) Characterization of CD201 + and CD34 + cells. ( C ) Representative flow cytometric analysis. ( D ) Quantification of percentages of CD34 + and CD201 + CD34 - within CD45 - CD31 - TSLP-tdTomato + stromal cells. ( E ) Quantitation of percentage of TSLP-tdTomato + cells among CD45 - cells in jejunal ( left ) and ileal ( right ) LP after 3 daily injections of GLP-2[Gly2]. ( F ) Intracellular antibody staining for TSLP protein in purified CD45 - CD31 - TSLP-tdTomato + stromal cells at 4 hr after incubation with 10 µg/ml GLP-2[Gly2] with Brefeldin A in vitro . ( G ) ELISA for TSLP protein recovered from ileal tissue homogenates from fasted Glp2r -/- mice after food gavage at 2hr ( H )( I ) Flow cytometric analysis of GATA3 + ILC2s in jejunal LP after 3 daily injections of GLP-2[Gly2]. ( H ) Quantification of percentage GATA3 + ILC2s among Lin - CD45 + cells. ( I ) Quantification of percentage of Ki67 + cells among GATA3 + ILC2s. ILC2s were gated on CD45 + Lin - GATA3 + cells. All data are biological replicates, n≥3. Data are representative of at least two independent experiments. Error bars indicate samples mean± SEM. *P<0.03.

Article Snippet: Mouse TSLP antibody for flow cytometric analysis was labeled using Alexa FluorTM 488 Antibody Labeling Kit (ThermoFisher, Cat# A20181) following the manufacture protocol.

Techniques: Quantitation Assay, Staining, Purification, Incubation, In Vitro, Enzyme-linked Immunosorbent Assay