Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

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 + and stromal populations in jejunal and ileal lamina propria in Flare-TSLP or non-reporter control (ctrl) mice. b , CD45 + cells were CD31 + endothelial cells. CD45 − CD31 − stromal cells were PDGFRa + 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 + and LYVE1 + consistent with lymphatic endothelial cells (LECs). 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 calcipotriol (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 or on day 7 and 12 post N. brasiliensis infection in Flare-TSLP or non-reporter mice. d.p.i., day post infection. g-h , Representative imaging of jejunum in Flare-TSLP or non-reporter mice on day 5 after N. brasiliensis ( g ) and in cecum day 21 after T. muris infection ( h ). Red, TSLP-tdTomato; Green, PDGFRa; White, EPCAM; Blue, DAPI. 20x objective. Scale bar, 100 µm. Inset scale bar, 50 µm. All data are biological replicates, n≥3. Data are representative of at least two independent experiments.

Article Snippet: Tslp , Mm00498739_m1.

Techniques: Imaging, Control, Expressing, Infection

Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

Figure Lengend Snippet: a , Gene targeting strategy for the flox-and-reporter of Tslp locus. Frt, target site for FLIPASE recombinase; pA, bovine growth hormone poly(A) tail; UTR, untranslated region. b , Representative imaging of jejunum (top) and ileum (bottom) of Flare-TSLP or nonreporter control mice. Red, TSLP-tdTomato; green, PDGFRα; white, EPCAM; blue, DAPI; ×20 objective. Scale bars, 50 µm (main images) and 20 µm (boxed areas I–IV). c , d , Flow cytometric analysis of stromal populations in jejunum and ileum siLP in Flare-TSLP mice. c , Percentage of total TSLP-tdTomato + cells in CD45 − population; N = 7 biological replicates each column group. d , Percentage of endothelial and nonendothelial stromal cells within TSLP-tdTomato + cells. Cells were stained with anti-CD31 and anti-PDPN antibodies; N = 3 biological replicates each column group. Error bars indicate samples mean ± s.e.m. e , RT–qPCR analysis of sorted CD45 − EPCAM + epithelial cells, CD45 − EPCAM + SiglecF + tuft cells, CD45 − CD31 − PDPN + fibroblasts, CD45 − CD31 − PDPN + TSLP-tdTomato + fibroblasts, and 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). Note that epithelial tuft cells can express Lgr5 -eGFP. Red, TSLP-tdTomato; green, Lgr5 -eGFP; white, EPCAM; blue, DAPI; ×20 objective. Scale bars, 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; ×20 objective. Scale bars, 100 µm. h , RT–qPCR analysis of sorted CD45 − CD31 − TSLP-tdTomato + CD201 + CD31 − , CD45 − CD31 − TSLP-tdTomato + CD34 + , CD45 − CD31 − CD201 + and CD45 − CD31 − CD34 + stromal subpopulations from siLP (whole tissue) of Flare-TSLP mice. Gene expression normalized to 18S.

Article Snippet: Tslp , Mm00498739_m1.

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

Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

Figure Lengend Snippet: a , Representative imaging of CD201 and CD34 expression in proximal and distal small intestine of Flare-TSLP; Lgr5 -eGFP dual reporter mice. Note that epithelial tuft cells can express Lgr5 -eGFP. Scale bar, 100 µm. b , Gating strategy for FACS sorting of CD45 − CD31 − TSLP-tdTomato + CD201 + , and CD45 − CD31 − TSLP-tdTomato + CD34 + cells from small intestinal LP (siLP, whole tissue) of Flare-TSLP mice. c , RT-qPCR analysis for Tslp, Foxl1, Wnt5a, Lgr5, Bmp7, Grem1, Rspo3, Wnt2b, Bmp4 expression in sorted CD45 − CD31 − TSLP-tdTomato + CD201 + , and CD45 − CD31 − TSLP-tdTomato + CD34 + , CD45 − CD31 − CD201 + , and CD45 − CD31 − CD34 + stromal cells from siLP (whole tissue) of Flare-TSLP mice. Data are biological replicates, n≥3. Data are representative of at least two independent experiments. Error bars indicate samples mean ± SEM.

Article Snippet: Tslp , Mm00498739_m1.

Techniques: Imaging, Expressing, Quantitative RT-PCR

Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

Figure Lengend Snippet: a , Schematic of the protocol for measuring intestinal TSLP after feeding. Mice were fasted for 16 h overnight 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 siLP by flow cytometric analysis after oral gavage at 2 h after overnight fasting. Biological replicates N = 19 for sham control group and N = 16 for refeeding group. Statistical analysis was performed using unpaired t -test, * P < 0.05. Error bars indicate samples mean ± s.e.m. c , ELISA of TSLP protein from supernatant of proximal jejunal tissue explants after oral gavage at 2 h in Pou2f3 − / − or WT mice after overnight fasting. Biological replicates with total N = 28, pooled from 2 independent experiments. Statistical analysis was performed using ANOVA with correction for multiple comparisons, * P < 0.05. Error bars indicate samples mean ± s.e.m. NS, nonsignificant. d , Schematic of the protocol for measuring siLP ILC2 activation after feeding. Mice were fasted for 16 h overnight and given access to standard chow and water ad libitum (refed) or maintained on water control (fasted). Tissues were harvested 4 h later. e , f , Percentage of IL-13 (Smart13) + ILC2 among ILC2s in proximal jejunal LP in Tslpr + /+ Red5Smart13 or Tslpr − / − Red5Smart13 mice after 16-h fasting followed by 4-h refeeding ad libitum. e , Representative flowplots. f , Quantification. ILC2s were gated as Lin − CD45 + IL-5 (Red5) + cells. Biological replicates with total N = 31, pooled from at least 2 independent experiments. Statistical analysis was performed using ANOVA with correction for multiple comparisons, ** P < 0.01, *** P < 0.001. Error bars indicate samples mean ± s.e.m. g , Percentage of IL-13 (Smart13) + ILC2s among ILC2s in proximal jejunal LP in Tslpr fl/fl Il5 Cre+ (Red5)Smart13 mice after 16-h fasting followed by 4-h water or refeeding ad libitum. ILC2s were gated as Lin − CD45 + IL-5 (Red5) + cells. Biological replicates N = 8 each column group, pooled from 2 independent experiments. Statistical analysis was performed using unpaired t -test. Error bars indicate samples mean ± s.e.m. h , i , Percentage of IL-13 (Smart13) + ILC2s among total ILC2s in proximal jejunal LP 4 h after refeeding ad libitum in overnight fasted Tslp fl/fl Pdgfrα CreERT2+ Smart13 or littermate control Pdgfrα +/+ Smart13 mice (ctrl) post-tamoxifen. h , Representative flowplots. i , Quantitation. ILC2s were gated on Lin − CD45 + GATA3 + cells. Biological replicates N = 6 for control group and N = 13 for experiment group, pooled from at least 2 independent experiments. Statistical analysis was performed using unpaired t -test, * P < 0.05. Error bars indicate samples mean ± s.e.m. j , Percentage of IL-13 (Smart13) + ILC2s among ILC2s in proximal jejunal LP 4 h after water or refeeding ad libitum in overnight fasted Tslp fl/fl Lyve Cre+ Smart13 mice. ILC2s were gated on Lin − CD45 + GATA3 + cells. Biological replicates N = 4 in each column group. Statistical analysis was performed using unpaired t -test, * P < 0.05. Error bars indicate samples mean ± s.e.m. Illustrations in a created with BioRender.com .

Article Snippet: Tslp , Mm00498739_m1.

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

Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

Figure Lengend Snippet: a-e , Feeding increases intestinal TSLP. a , Percentage of TSLP-tdTomato + cells among CD45 − cells in ileal lamina propria by flow cytometric analysis in fasted mice 2 hr after oral gavage of food (refed) or water as volumetric control (sham). Biological replicates N = 20 for control group and N = 17 for refeeding group. Statistic analysis was performed using unpaired t test, **P < 0.01. Error bars indicate samples mean ± SEM. b , ELISA of TSLP protein recovered from proximal jejumal tissue homogenates after oral gavage of food at designated times in fasted wild type (WT) mice. Biological replicates N = 3 per time point. Error bars indicate samples mean± SEM. c , ELISA of TSLP protein recovered from distal ileal tissue homogenates from fasted WT mice 2 hr after oral food gavage. Biological replicates N = 3 - 4 per time point. Statistical analysis was performed using ANOVA with correction for multiple comparisons. Error bars indicate samples mean ≥ SEM. d , ELISA of TSLP protein recovered from proximal jejunal tissue homogenates after oral gavage 2 hr in Pou2f3 − / − or WT mice after overnight fasting. Biological replicates N = 3 – 7 each column group. Error bars indicate samples mean ± SEM. e , ELISA of TSLP protein recovered from distal ileal tissue homogenates after oral gavage 2 hr in Pou2f3 − / − or WT mice after overnight fasting. Biological replicates N = 3 - 4 each column group. Error bars indicate samples mean ± SEM.

Article Snippet: Tslp , Mm00498739_m1.

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

Figure Lengend Snippet: a , Gating strategy for FACS sorting of TSLP- tdTomato + cells from intestinal LP of Flare-TSLP mice. b-f , scRNA-seq analysis of pooled proximal and distal siLP TSLP- tdTomato + cells. b , Expression levels of marker genes for annotated cell types. c , Feature plots represent marker genes for telocytes, VTTs, SEMFs, trophocytes and universal fibroblasts/mesothelial-like cells. d , Expression levels of WNT and BMP signaling molecules for annotated cell types. e , Heatmap representation of top 20 most significantly changed genes including both directions from differential gene expression (DE) analysis between cell types in the proximal and distal small instestine. Columns were manually ordered by cell type. Plot shows z-scores. f , Expression levels of gut hormone receptors in Foxl1+ telocytes.

Article Snippet: Tslp , Mm00498739_m1.

Techniques: Expressing, Marker, Gene Expression

Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

Figure Lengend Snippet: a – c , scRNA-seq analysis of sorted TSLP-tdTomato + cells from the siLP. a , UMAP representing cell clustering analysis. b , Expression of gut hormorne receptors by TSLP-tdTomato + stromal cells. c , Feature plots representing Glp1r (top) and Glp2r (bottom) expression. d , RT–qPCR analysis of Glp1r (left) and Glp2r (right) expression on sorted gut. CD45 − EPCAM + (epithelial cells), tuft cells, CD45 + PDPN − (hematopoietic cells), CD45 − CD31 + PDPN + (LECs), CD45 − CD31 − TSLP-tdTomato + total stromal cells, CD45 − CD31 − TSLP-tdTomato + CD201 + (telocytes) and CD45 − CD31 − TSLP-tdTomato + CD34 + (trophocytes). Biological replicates N = 3 or 4, pooled from 2 independent experiments. Error bars indicate samples mean ± s.e.m. e , Percentage of TSLP-tdTomato + cells among CD45 − cells in proximal jejunal LP after three consecutive GLP-2[Gly2] s.c. injections as assessed by flow cytometric analysis. Biological replicates N = 9 for each column group, pooled from at least 2 independent experiments. Statistical analysis was performed using unpaired t -test, ** P < 0.01. Error bars indicate samples mean ± s.e.m. f , Flow cytometric analysis showing percentage of total TSLP-tdTomato + cells among CD45 − cells in proximal jejunal LP from fasted Glp2r − / − Flare-TSLP mice after oral food gavage at 2 h. Biological replicates N = 7 for control group and N = 9 for refeeding group, pooled from at least 2 independent experiments. Statistical analysis was performed using unpaired t -test. Error bars indicate samples mean ± s.e.m. g , ELISA of TSLP protein from supernatant of proximal jejunal tissue explants after oral gavage at 2 h in Glp2r − / − or WT mice after overnight fasting. Biological replicates with total N = 25, pooled from at least 2 independent experiments. Statistical analysis was performed using ANOVA with correction for multiple comparisons, * P < 0.05, ** P < 0.01. Error bars indicate samples mean ± s.e.m. h , Quantification of percentage of IL-13 (Smart13) + ILC2s among proximal jejunal LP ILC2s from fasted Glp2r − / − Smart13 mice after 16-h fasting followed by 4-h water or refeeding ad libitum. ILC2s were gated on Lin − CD45 + GATA3 + cells. Biological replicates N = 5 for control group and N = 8 for refeeding group, pooled from at least 2 independent experiments. Statistical analysis was performed using unpaired t -test. Error bars indicate samples mean ± s.e.m. i , Quantification of percentage of IL-13 (Smart13) + ILC2s among proximal jejunal LP ILC2s in Tslpr +/+ Red5Smart13 or Tslpr − / − Red5Smart13 mice after three daily injections of GLP-2[Gly2]. ILC2s were gated on Lin − CD45 + IL-5(Red5) + cells. Biological replicates with total N = 38, pooled from at least 2 independent experiments. Statistical analysis was performed using ANOVA with correction for multiple comparisons, * P < 0.05, **** P < 0.0001. Error bars indicate samples mean ± s.e.m. j , Quantification of percentage of IL-13 (Smart13) + ILC2s among proximal jejunal LP ILC2s in Tslpr fl/fl Il5 Cre+ Smart13 after three daily injections of GLP-2[Gly2] or vehicle control. ILC2s were gated on Lin − CD45 + IL-5(Red5) + cells. Biological replicates N = 6 for vehicle control group and N = 9 for GLP-2[Gly2] injection group, pooled of 2 independent experiments. Statistical analysis was performed using unpaired t -test. Error bars indicate samples mean ± s.e.m. k , l , Flow cytometric analysis of percentage of IL-13 (Smart13) + ILC2s among proximal jejunal LP ILC2s in Tslp fl/fl Pdgfrα CreERT2+ Smart13 or littermate Tslp fl/ fl Smart13 controls post-tamoxifen followed by three daily injections of GLP-2[Gly2]. k , Representative flowplots. l , Quantitation. ILC2s were gated as Lin − CD45 + GATA3 + cells. Biological replicates N = 7 for each column group, pooled from at least 2 independent experiments. Statistical analysis was performed using unpaired t -test, ** P < 0.01. Error bars indicate samples mean ± s.e.m. m , Flow cytometric analysis of percentage of IL-13 (Smart13) + ILC2s among proximal jejunal LP ILC2s in Tslp fl/fl Nes CreERT2+ Smart13 or littermate Tslp fl/ fl Smart13 controls post-tamoxifen followed by three daily injections of GLP-2[Gly2]. Biological replicates N = 6 for control group and N = 5 for experiment group, pooled from 2 independent experiments. Statistical analysis was performed using unpaired t -test, * P < 0.05. Error bars indicate samples mean ± s.e.m.

Article Snippet: Tslp , Mm00498739_m1.

Techniques: Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Injection, Quantitation Assay

Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

Figure Lengend Snippet: a , UMAPs representing cell clustering in pooled cells (total intestine LP cells), siLP, LILP and cecum LP samples. Unsupervised cell clustering analysis revealed 17 clusters based on differential gene expression. Annotation was conducted according to dominant cluster-specific genes, while also considering existing stromal subsets. Subsets of fibroblasts were categorized as SEMFs (telocytes_1, telocytes_2, SEMFs_1, SEMFs_2), trophocytes (trophocytes_1, trophocytes_2, trophocytes_3), and recently identified Pi16 + universal fibroblasts or mesothelial-like cells (mesothelial-like_1, mesothelial-like_2). We also identified myofibroblasts (MFs), and an undefined cluster characterized by Pdgfra hi CD81 + Ptn + fibroblasts. Additional cells included LECs and pericytes. Some cell types were enriched in specific tissues; for instance, trophocytes, Pdgfra hi CD81 + cells, and LECs were primarily found in small intestine samples, while SEMFs_2 were more abundant in the large intestine and cecum but not in small intestine. Telocytes_2 were mostly enriched in the large intestine with few minimal in cecum and none in small intestine. b , Gut hormone receptor expression in total pooled intestine LP cells, LILP and cecum LP samples. c , Heatmap representing top 20 fold-change genes including both directions between tissue comparisons in DE analysis. Plot shows log2 normalized expression. The transcriptomic signatures of TSLP-tdTomato + cells were similar betwen proximal and distal small intestine, as well as between large intestine and cecum.

Article Snippet: Tslp , Mm00498739_m1.

Techniques: Gene Expression, Expressing

Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

Figure Lengend Snippet: a , Heatmaps representing expression levels of gut hormone receptors in PDGFRa hi telocytes , PDGFRa lo trophocytes , CD31 + LYVE1 − endothelial cells , LYVE1 + LECs , Foxl1 + telocytes and Foxl1 − stromal cells from mouse datasets. b-d , 10x Genomics published Visium mouse small intestine dataset . b , Hematoxylin and eosin staining of the intestinal tissue specimen. c , Color-coded cell clusters in the lamina propria (LP) shown in situ ( top ) and on a UMAP ( bottom ). d , UMAP feature plots representing gene expression level and distribution of Pdgfra , Lyve1 , Tslp and Glp2r . Tslp and Glp2r expression enriched in the stromal populations of intestinal LP. e-h , Analyses of human intestinal datasets , . e , UMAP representing cell clustering of human dataset . f , Feature plots of TSLP ( left ) and GLP2R ( right ) expression levels. g , UMAP representing cell clustering of human dataset . h , Feature plots of TSLP ( left ) and GLP2R ( right ) expression levels.

Article Snippet: Tslp , Mm00498739_m1.

Techniques: Expressing, Staining, In Situ, Gene Expression

Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

Figure Lengend Snippet: a , Flow cytometric analysis of TSLP-tdTomato + cells in ileal lamina propria (LP) after 3 daily GLP-2[Gly2] injections. Biological replicates N = 4-5 for each group. b-e Flow cytometric analysis of TSLP-tdTomato + cells in jejunal and ileal lamina propria (LP) at 2 hr after GLP-2[Gly2] administration. b , Percentage of total TSLP-tdTomato + cells among CD45 − cells in jejunal ( left ) and ileal LP ( right ). Biological replicates N = 7-10 (left) and N = 8-9 (right) for each group. c , Proportion of CD31 + PDPN + LECs, CD31 − PDPN + and CD31 − PDPN lo stromal populations within TSLP-tdTomato + cells in jejunum ( left ) and in ileum ( right ). Biological replicates N = 3-4 each group. d-e , Characterization of CD201 + and CD34 + cells within CD45 − PDPN + CD31 − TSLP-tdTomato + stromal cells. d , Representative flow cytometric analysis. e , Quantification. Biological replicates N = 3-4 each column group. f , Intracellular antibody staining for TSLP protein in sorted CD45 − CD31 − TSLP-tdTomato + stromal cells from small intestinal LP 4 hr after incubation with 10 µg/ml GLP-2[Gly2] with Brefeldin A in vitro . g-h , Mouse fibroblasts treated with agonists ( g ) and/or inhibitors ( h ). TSLP levels were assessed by flow cytometric analysis using frequency of TSLP + cells among total cultured fibroblasts characterized by CD45 − PDPN + . Biological replicates N = 7 each group. i , Mouse intestinal explants treated with agonists and/or inhibitors. TSLP protein levels in the supernatants were measured by ELISA. Biological replicates total N = 77. j , TSLP protein levels in supernatants of human fibroblast cultures treated with agonists and/or inhibitors. Paired t-tests were performed for statistical analysis. Biological replicates N = 3. k , TSLP protein recovered from jejunal and ileal tissue homogenates from fasted Glp2r − / − mice 2 hr after food gavage. Biological replicates N = 3-4 each group. l-m , Flow cytometric analysis of ILC2s in jejunal LP in Tslpr ± + and Tslpr − / − mice after 3 daily injections of GLP-2[Gly2]. l , Percentage of ILC2s among Lin − CD45 + cells. m , Quantification of percentage of Ki67 + cells among ILC2s. ILC2s were gated on CD45 + Lin − GATA3 + cells. Biological replicates N = 9-14 for each group. Graphs represent data pooled from at least two experiments. Error bars indicate samples mean± SEM. Unless specified, upaired t-test was used for analyses of difference between 2 groups. ANOVA test was used for analyzing experiments with >2 groups adjusted for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Tslp , Mm00498739_m1.

Techniques: Staining, Incubation, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

Figure Lengend Snippet: a-c , Flow cytometric analysis of ILC2s in large intestinal lamina propria (LILP) on day 21 post T. muris infection. Percentage of IL-13 (Smart13) + ILC2 among ILC2s in LI LP in Tslpr − / − or Tslpr ± + mice, biological replicates N = 4-8 each column group, ****P < 0.0001 ( a ), Tslpr fl/fl Il5 Cre+ or WT controls, biological replicates N = 3-10 each column group, *P < 0.05, ***P < 0.001 ( b ), and Tslp fl/fl Pdgfra CreERT2+ or Tslp fl/fl controls, biological replicates N = 3-12 each column group, **P < 0.01 ( c ). ILC2s were gated on Lin − CD45 + GATA3 + cells. d , RT-qPCR analysis for Nes expression in sorted cells from the small intestine, including CD45 − EPCAM + epithelial cells, tuft cells, CD45 + PDPN − hematopoietic cells, CD45 − CD31 + PDPN + LECs, CD45 − CD31 − PDPN + , CD45 − CD31 − CD201 + , and CD45 − CD31 − CD34 + stromal cells. Biological replicates N = 3-4 each column group. e-f , Flow cytometric analyses of LILP ILC2s in Tslp fl/fl Nes CreERT2+ or control mice on 21 d.p.i. Percentage of IL-13 (Smart13) + ILC2 among ILC2s in LI LP in Tslp fl/fl Nes CreERT2+ or control ( e ) and the expression levels of ILC2s activation markers including HuCD4 (Smart13) and ICOS were lower in Tslp fl/fl Nes CreERT2+ mice than controls ( f ). ILC2s were gated on Lin − CD45 + GATA3 + cells. Biological replicates N = 3-9 each column group, **P < 0.01, ***P < 0.001. g-h , Flow cytometric analysis of LILP ILC2s in Glp2r − / − and Glp2r ± + mice 21 d.p.i. Flow charts represent IL-13 (Smart13) levels ( g ) and percentage of IL-13 (Smart13) + ILC2s among total ILC2s in LI LP ( h ). ILC2s were gated on Lin − CD45 + GATA3 + cells. Biological replicates N = 4-11 each column group, *P < 0.05, **P < 0.01. Data are representative of at least two independent experiments. Statistical analyses were performed using ANOVA with correction for multiple comparisons. Error bars indicate samples mean ± SEM.

Article Snippet: Tslp , Mm00498739_m1.

Techniques: Infection, Quantitative RT-PCR, Expressing, Control, Activation Assay

Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

Figure Lengend Snippet: a , Representative imaging of tuft cells in jejunum after three daily injections of GLP-2[Gly2]. EdU was injected 24 h before tissue harvest. Green, EdU; red, DCLK1; white, EPCAM; blue, DAPI; ×20 objective. Scale bars, 100 µm. b , Quantification of jejunal tuft cells after three daily GLP-2[Gly2] injections or vehicle control in WT, Tslpr −/− , Tslpr fl/fl Il5 Cre+ , Il4rα −/− , Il13rα −/− and Il4rα fl/fl Vil1 Cre+ mice. For tuft cell quantification, images were acquired using ×20 objective, and total DCLK1 + EPCAM + cells in each field were counted and normalized by total number of villus–crypt axes in the field. Each dot represents an imaging field, total N = 232; data pooled from 3 to 10 mice. A plot with averages of each mouse is included in Extended Data Fig. . Statistical analysis was performed using ANOVA with correction for multiple comparisons, **** P < 0.0001. Error bars indicate samples mean ± s.e.m. c , Quantification of jejunal tuft cells after three daily GLP-2[Gly2] or vehicle control injections in Tslp fl/fl , Tslp fl/fl Nes CreERT2+ and Tslp fl/fl Pdgfrα CreERT2+ mice. Each dot represents an imaging field, total N = 80; data pooled from 3 to 6 mice. A plot with averages of each mouse is included in Extended Data Fig. . Statistical analysis was performed using ANOVA with correction for multiple comparisons, **** P < 0.0001. Error bars indicate samples mean ± s.e.m. d , Representative imaging showing CCK + EECs in Vil1 Flp Cck Cre R26 Dual − hM3Dq mice. Red, Cck -mCherry; green, Vil1-GFP; blue, DAPI; ×20 objective. Scale bar, 100 µm. e – g , Quantification of jejunal tuft cells after administration of clozapine N-oxide CNO or vehicle control in Vil1 Flp Cck Cre R26 Dual − hM3Dq mice. e , Percentage of tuft cells among the CD45 − epithelial fraction by flow cytometric analysis. Two independent experiment repeats were combined to generate the plot. Biological replicates N = 6 for each column group were compared using two-tailed unpaired t -test, ** P < 0.005. Error bars indicate samples mean ± s.e.m. f , Representative imaging of tuft cells. Red, DCLK1; white, Vil1-GFP; blue, DAPI; ×20 objective. Scale bars, 100 µm. g , Quantification of tuft cells. Each dot represents an imaging field, N = 25 for control group and N = 28 for CNO treatment group; data pooled from 6 mice each group from 2 independent experiments. A plot with averages of each mouse is included in Extended Data Fig. . Statistical analysis was performed using two-tailed unpaired t -test, **** P < 0.0001. Error bars indicate samples mean ± s.e.m.

Article Snippet: Tslp , Mm00498739_m1.

Techniques: Imaging, Injection, Control, Two Tailed Test

Journal: Nature Immunology

Article Title: TSLP links intestinal nutrient sensing with amplification of the ILC2–tuft cell circuit

doi: 10.1038/s41590-025-02328-y

Figure Lengend Snippet: a , Representative imaging of jejunal tuft cells in Tslpr −/− , Tslpr fl/fl Il5 Cre+ , Il4ra −/− , Il13ra1 −/− and Il4ra fl/fl Vil1 Cre+ mice. Green, EdU; Red, DCLK1; White, EPCAM; Blue, DAPI. 20x objective. Scale bar, 100 µm. b , Quantification of jejunal tuft cells after 3 daily GLP-2[Gly2] injections in WT, Tslpr −/− , Tslpr fl/fl Il5 Cre+ , Il4ra −/− , Il13ra −/− and Il4ra fl/fl Vil1 Cre+ mice. For tuft cell quantification, images were acquired using 20x objective and total DCLK1 + EPCAM + cells in each field were counted and normalized by total number of villus-crypt axes in the field. Each dot represents a mouse, N = 3-10 each column. Statistical analysis was performed using ANOVA with correction for multiple comparisons, ****P < 0.0001. Error bars indicate samples mean± SEM. c , Representative imaging of jejunal tuft cells in Tslp fl/fl , Tslp fl/fl Nes CreERT2+ , and Tslp fl/fl Pdgfra CreERT2+ mice. Green, EdU; Red, DCLK1; White, EPCAM; Blue, DAPI. 20x objective. Scale bar, 100 µm. d , Quantification of jejunal tuft cells after 3 daily GLP-2[Gly2] injections in Tslp fl/fl , Tslp fl/fl Nes CreERT2+ , and Tslp fl/fl Pdgfra CreERT2+ mice. Each dot represents a mouse, N = 3-6. Statistical analysis was performed using ANOVA with correction for multiple comparisons, *P < 0.05, **P < 0.01. Error bars indicate samples mean± SEM. e , Schematic illustrating the Vil1 Flp Cck Cre R26 Dual − hM3Dq DREADD and CNO system. f-g , Quantification of jejunal tuft cells in Vil1 Flp Cck Cre R26 Dual − hM3Dq mice after administration of clozapine N-oxide (CNO) or vehicle control. Each dot represents a mouse. f , Representative imaging of tuft cells. Red, DCLK1; White, Vil1 -GFP; Blue, DAPI. 20x objective. Scale bar, 100 µm. g , Quantification of tuft cells. Two experiment repeats, each dot represents a mouse, N = 6 for each column group. Statistical analysis was performed using two-tailed unpaired t-test,*P < 0.05. Error bars indicate samples mean± SEM. h , Proposed model for the mechanism of GLP-2 – TSLP – ILC2 signaling pathway in promoting intestine tissue remodeling. GLP-2 from L cells ( 1 ) stimulates telocytes to produce TSLP ( 2 ) that activates ILC2s ( 3 ), thus linking nutrient ingestion and IL-13-mediated amplification of epithelial tuft cells. ( 4 ) N. brasiliensis short circuits the physiologic pathway by directly stimulating tuft cells to release IL-25 that activates ILC2s and drives the ILC2–tuft cell circuit amplification. Panels e and h created using BioRender.com .

Article Snippet: Tslp , Mm00498739_m1.

Techniques: Imaging, Control, Two Tailed Test, Amplification