Tachykinin receptor 2 tissue expression

a, Fluorescent image of a transgenic C. elegans expressing npr-22::GFP under the control of the endogenous npr-22 promoter. GFP expression was detected in the intestinal cells and in several pairs of neurons in the head. A, anterior; P, posterior; V, ventral; D, dorsal. Scale bar, 50 μm. b, UMAP visualization depicting gene clusters based on mRNA expression profiles from The Human Protein Atlas44 (HPA). The cluster containing hTacr2 (cluster 45, comprising 300 genes) is highlighted in blue. Functional annotation provided by HPA characterizes the shared specificity (Intestine) and biological function (Digestion) of genes within this cluster. c, The gene expression levels of Tacr1, Tacr2, and Tacr3 were assessed throughout the entire mouse small intestine using qPCR. The intestine was divided into eight equal segments, each measuring approximately 4-5 cm. Actb was used as the housekeeping gene. Data are presented as the fold change relative to the expression level of Tacr2 in the first segment ± SEM. n = 6 biological replicates. d, Fluorescent image of mouse jejunum stained with NK2R antibody (green) and DAPI (blue). Scale bar: 20 μm. Inset: magnified view of the region enclosed by the white line in the fluorescent image. The jejunum was harvested from male C57BL6/J mice (8 weeks old, low-fat diet). e, The strategy of CRISPR-Cas9 mediated genome editing, depicting the genomic region of Tacr2 and the locations of the Cas9 cutting sites for Tacr2-/-. The deleted exon 2 is marked in red. f, qPCR analysis of Tacr2 mRNA expression in intestinal epithelial RNA isolated from male and female control and Tacr2-/- mice. n = 7 male control, n = 6 female control, n = 6 male Tacr2-/-, n= 6 female Tacr2-/-. g,j, Body weights of male (g) and female (j) Tacr2-/- and control mice (8-12 weeks old) were monitored weekly over a 10-week period while maintained on a low-fat diet (LFD). n = 8 control and n = 12 Tacr2-/-mice (g); n = 11 control and n = 13 Tacr2-/- mice (j). h,k, Following an overnight fast, male (h) and female (k) mice were re-fed with either LFD or HFD, and cumulative food intake was measured over a 4 hr refeeding period. n = 7 control (LFD), n = 7 Tacr2-/- (LFD), n = 7 control (HFD), and n = 8 Tacr2-/- (HFD) mice (h); n = 6 per group (k). i,l, Glucose tolerance tests were performed in week 10 in male (i) and female (l) mice. n = 5 control and n = 6 Tacr2-/-mice per group. Data was analyzed using an unpaired Student’s t-test (two-tailed) (f,h,k) or two-way ANOVA with repeated measures (g,i,j,l). Data are shown as mean ± SEM. nsp>0.05. Black bars/lines represent control mice; red bars/lines represent Tacr2-/- mice.

Sex-specific transcriptional responses to high-fat diet in Tacr2-/- mice

a, Schematic of the experimental workflow. Control and Tacr2-/- mice were fed a low-fat diet (LFD) or high-fat diet (HFD) for 1 week before sacrifice. Following tissue collection, the upper intestinal epithelial layer was isolated, and total RNA was extracted for subsequent bulk RNA sequencing. Panel created with BioRender.com/17nczm5. b, Principal component analysis (PCA) of the RNA-Seq data from male control and Tacr2-/-mice on LFD or HFD. n = 3 control (LFD), n = 3 control (HFD), n = 4 Tacr2-/-(LFD), n = 3 Tacr2-/- (HFD). c,d, Volcano plots of differentially expressed genes in male Tacr2-/- versus control mice under LFD (c) or HFD (d) conditions. e-h, Gene ontology (GO) enrichment analyses of genes differentially expressed in male Tacr2-/- mice versus controls on LFD (e, upregulated; f, downregulated) or HFD (g, upregulated; h, downregulated). Bubble size indicates the number of genes associated with each GO term, and color represents the false discovery rate (FDR). i, Principal component analysis of RNA-Seq data from female control and Tacr2-/- mice on LFD or HFD. n = 4 control (LFD), n = 4 control (HFD), n = 3 Tacr2-/-(LFD), n = 3 Tacr2-/- (HFD). j,k, Volcano plots of differentially expressed genes in female Tacr2-/- versus control mice under LFD (j) or HFD (k) conditions. l-o, Gene ontology enrichment analyses of genes differentially expressed in female Tacr2-/-mice versus controls on LFD (l, upregulated; m, downregulated) or HFD (n, upregulated; o, downregulated). Bubble size and color reflect gene count and FDR, respectively. p,q, Venn diagrams showing overlap of differentially expressed genes (adjusted p-value < 0.05 and |log2 fold change| > 1.0) between LFD and HFD conditions in male (p) and female (q) datasets. Numbers indicate the number of shared or unique genes between conditions.

NK2R inhibition attenuates DSS-induced weight loss and intestinal inflammation in male mice

a, Bulk RNA-Seq data were integrated with publicly available single-cell RNA-Seq datasets to estimate cell-type composition in the small intestine of male mice. Differences in estimated cell-type proportions were analyzed based on diet (D), genotype (G), or their interaction (I). b, Schematic representation of the experimental workflow for the DSS-induced colitis model. Male and female mice received DSS solution (3% for males, 5% for females) or vehicle (drinking water) ad libitum for 7 consecutive days. Following treatment, tissues were collected for downstream analyses or subjected to an in vivo gut permeability assay. Panel created with BioRender.com/r5jdhzu. c, Daily monitoring of body weights in male control and Tacr2-/- mice treated with DSS or vehicle over a 7-day period. n = 5 control (H2O), n = 5 control (DSS), n = 6 Tacr2-/-(H2O), and n = 6 Tacr2-/- (DSS). d, Comparison of body weight changes from baseline (day 0) to day 7, derived from the data shown in panels (c). e,f, Lipocalin-2 (LCN2) concentrations in blood (e) and feces (f) from male control and Tacr2-/-mice after 7 days of DSS or vehicle treatment, as determined by ELISA. n = 6 control (H2O), n = 5 control (DSS), n = 4 Tacr2-/- (H2O), and n = 4 Tacr2-/- (DSS) for (e); n = 5 control (H2O), n = 5 control (DSS), n = 5 Tacr2-/- (H2O), and n = 4 Tacr2-/- (DSS) for (f). g, Colon tissues from male control and Tacr2-/-mice treated with DSS or vehicle for 7 days were collected, sectioned, and stained with hematoxylin and eosin (H&E). Representative images of stained colon sections are shown. h, Representative histological images (g) were evaluated and scored according to the degree of tissue damage. n = 5 control (H2O), n = 4 control (DSS), n = 6 Tacr2-/- (H2O), and n = 4 Tacr2-/-(DSS). i, Gut permeability in male control and Tacr2-/-mice following 7 days of DSS or vehicle treatment was assessed by oral gavage of 4 kDa FITC-dextran. Blood was collected 4 hours post-gavage, and FITC fluorescence was measured in plasma to quantify gut barrier integrity. n = 10 control (H2O), n = 9 control (DSS), n = 9 Tacr2-/- (H2O), and n = 8 Tacr2-/- (DSS). j, Daily monitoring of body weights in male wild-type mice treated with DSS or vehicle for 7 days, in combination with daily injections of the NK2R antagonist GR159897 (2.5 mg/kg) or vehicle (PBS). DSS administration was discontinued on day 7. n = 8 (H2O+Vehicle), n = 8 (DSS+Vehicle), n = 7 (DSS+GR159897). k, Comparison of body weight changes from baseline (day 0) to day 8, derived from the data shown in panel (j). l,m, Lipocalin-2 (LCN2) concentrations in blood (l) and feces (m) from male wild-type mice with indicated treatment on day 8, as determined by ELISA. n = 8 (H2O+Vehicle), n = 7 (DSS+Vehicle), and n = 8 (DSS+GR159897) for (l); n = 7 (H2O+Vehicle), n = 8 (DSS+Vehicle), and n = 8 (DSS+GR159897) for (m). n, Colon tissues from male wild-type mice with indicated treatment on day 8 were collected, sectioned, and stained with hematoxylin and eosin (H&E). Representative histological images were evaluated and scored according to the degree of tissue damage. n = 8 (H2O+Vehicle), n = 8 (DSS+Vehicle), and n = 8 (DSS+GR159897). Statistical analysis was performed using one-way or two-way ANOVA followed by Holm-Sidak post hoc tests. Data are shown as mean ± SEM, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Black bars/lines represent control mice; red bars/lines represent Tacr2-/- or GR159897-treated mice.

Tacr2-/-mice exhibit enhanced intestinal lipid absorption

a, Heat maps display Z-scores of normalized expression levels for genes encoding lipid droplet-associated proteins in male mice across experimental conditions. Hierarchical clustering of individual samples (top dendrogram) and genes (left dendrogram) is shown, with branch lengths reflecting the similarity between clusters. Wild-type control (WT) samples are labeled in black text, Tacr2-/-(KO) samples in red text, and samples from mice on a high-fat diet (HFD) are indicated in bold. b, Schematic representation of the experimental workflow for assessing intestinal lipid absorption. Mice were administered an oral gavage of oil containing either 14C-triolein (c,f) or C16-BODIPY (d,e,g,h), followed by quantification of lipids in intestinal tissue and blood. Panel created with BioRender.com/bb165i5. c,f, Oral lipid tolerance test (OLTT) was performed in male (c) and female (f) control and Tacr2-/- mice. Blood lipid levels were measured over a 4-hour period following oral gavage and expressed as disintegrations per minute (DPM) per μL of plasma. n = 8 control males, n = 7 Tacr2-/-males for (c); n = 7 control females, n = 9 Tacr2-/- females for (f). d,g, BODIPY fluorescence was measured in intestinal sections from male (d) and female (g) control and Tacr2-/-mice following oral gavage with oil containing C16-BODIPY. n = 5 control males, n = 5 Tacr2-/- males (d); n = 6 control females, n = 7 Tacr2-/- females (g). e,h, Representative fluorescence images of intestinal sections from male (e) and female (h) mice corresponding to the samples shown in panels (d) and (g), respectively. i, Triglyceride levels were measured in each plasma fraction from wildtype mice fed a HFD for 10 weeks and pretreated with LPL inhibitor and either vehicle, the NK2R agonist GR64349, or NK2R antagonist GR159897 prior to oral oil gavage. n=8 mice per treatment group. j, Representative images and quantification of Oil Red O (ORO)-stained intestinal sections from male and female control and Tacr2-/- mice following 12 weeks of HFD feeding. n = 4 control males, n = 4 Tacr2-/- males, n = 4 control females, n = 4 Tacr2-/-females. k, Schematic representation of the experimental workflow for assessing the effects of NK2R agonism on body mass. Diet-induced obese (DIO) wild-type mice were administered daily intraperitoneal injections of EB1002 or vehicle for 21 consecutive days. Panel created with BioRender.com/3cj6oym. l,m, Body weights (l) and food intake (m) were monitored over a 3-week period in lean and diet-induced obese (DIO) mice treated with vehicle, high-dose EB1002 (1000 nmol), or low-dose EB1002 (300 nmol). n = 8 (lean: vehicle), n = 7 (DIO: vehicle), n = 8 (DIO: EB1002, 1000nmol) and n = 8 (DIO: EB1002, 300nmol). n, The fasted blood glucose levels were determined in lean and diet-induced obese (DIO) mice treated with vehicle or high-dose EB1002 (1000 nmol). n = 4 (lean: vehicle), n = 4 (DIO: vehicle), and n = 4 (DIO: EB1002, 1000nmol). o, Lean mass and fat mass were measured over 3 weeks in lean and diet-induced obese (DIO) mice treated with vehicle, high-dose EB1002 (1000 nmol), or low-dose EB1002 (300 nmol). n = 8 (lean: vehicle), n = 7 (DIO: vehicle), n = 8 (DIO: EB1002, 1000nmol) and n = 8 (DIO: EB1002, 300nmol). Statistical analyses were performed using two-way ANOVA with repeated measures (c,d,f,g,l,m), unpaired two-tailed Student’s t-test (b,j), one-way ANOVA with Holm-Sidak post hoc analysis (n), or two-way ANOVA with Holm-Sidak post hoc analysis (o). Data are presented as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, nsp>0.05. Black bars/lines indicate control mice, red bars/lines indicate Tacr2-/- mice and blue bars/lines indicate NK2R agonist-treated mice.

16S rRNA sequencing analysis of gut microbial populations

a, Shannon index (α-diversity) of fecal microbial populations in control and Tacr2-/- mice fed a LFD or HFD. n = 7 control (LFD), n = 7 control (HFD), n = 8 Tacr2-/- (LFD), n = 8 Tacr2-/-(HFD). Data were analyzed using two-way ANOVA followed by Holm-Sidak’s post hoc analysis; genotype effect: p = 0.039, diet effect: ns, genotype x diet interaction: ns. b, Principal Coordinate Analysis (PCoA) plot based on weighted UniFrac distances illustrating β-diversity of microbial communities in Tacr2-/- and control mice fed a LFD or HFD. Each point represents a microbial community from an individual sample, color-coded by genotype (Tacr2-/-, red; control, black) and diet (LFD, open circles; HFD, closed circles). c, Microbial populations were sorted at the phylum level across experimental conditions. Firmicutes/Bacteroidetes (F/B) ratio was calculated in control and Tacr2-/- mice fed a LFD or HFD. n = 7 control (LFD), n = 8 Tacr2-/- (LFD), n = 7 control (HFD), n = 8 Tacr2-/-(HFD). Data were analyzed using two-way ANOVA followed by Holm-Sidak post hoc analysis. Data are shown as mean ± SEM; **p<0.01, *p<0.05. d,e, Operational Taxonomic Units (OTUs) were analyzed using Linear Discriminant Effect Size (LEfSe) to identify taxa differentially enriched by diet or genotype. d, Bar graph showing Linear Discriminant Analysis (LDA) scores for 66 diet-dependent OTUs (34 associated with LFD, 32 associated with HFD). e, Bar graph showing LDA scores for 104 genotype-dependent OTUs (60 associated with control mice, 44 associated in Tacr2-/- mice). f, Relative abundances of 18 OTUs shared between the diet- and genotype-dependent analyses. OTUs were filtered using a Linear Discriminant Analysis (LDA) score threshold > 2.0. In panels (d) and (e), OTUs shared between diet and genotype analyses are indicated in green text. Schematic in panel created with BioRender.com/o7o48zt.

Tacr1 and Tacr3 expression in Tacr2-/- male mice.

a, UMAP visualization depicting gene clusters based on mRNA expression profiles from The Human Protein Atlas44 (HPA). The clusters containing hTacr1 (cluster 62, comprising 389 genes) and hTacr3 (cluster 76, comprising 175 genes) are highlighted. Functional annotation provided by HPA characterizes the shared specificity (Connective tissue or Retina) and biological function (ECM organization or Visual perception) of genes within the clusters. b, qPCR analysis of Tacr1 and Tacr3 mRNA expression in intestinal epithelial RNA extracted from male control and Tacr2-/- mice. n = 5 control and n = 4 Tacr2-/- mice for Tacr1, n = 5 per group for Tacr3.

Tacr2-/-mice show no significant changes in body weight compared to wild-type controls under acute high-fat diet (HFD) challenge.

a,b, Body weight changes from baseline (day 0) to day 7 in male (a) and female (b) control and Tacr2-/- mice fed a low-fat diet (LFD) or high-fat diet (HFD) for one week. n = 8 control (LFD), n = 12 Tacr2-/- (LFD), n = 7 control (HFD), and n = 9 Tacr2-/- (HFD) for (a); n = 11 control (LFD), n = 13 Tacr2-/-(LFD), n = 8 control (HFD), and n = 9 Tacr2-/- (HFD) for (b). Data were analyzed using two-way ANOVA with Holm-Sidak post hoc tests. Data are presented as mean ± SEM. nsp > 0.05. Black bars indicate control mice; red bars indicate Tacr2-/- mice.

NK2R inhibition does not suppress DSS-induced weight loss and intestinal inflammation in female mice.

a, Bulk RNA-Seq data were integrated with publicly available single-cell RNA-Seq datasets to estimate cell-type composition in the small intestine of female mice. Differences in estimated cell-type proportions were analyzed based on diet (D), genotype (G), or their interaction (I). b, Daily monitoring of body weights in female control and Tacr2-/- mice treated with DSS or vehicle over a 7-day period. n = 10 control (H2O), n = 9 control (DSS), n = 9 Tacr2-/-(H2O), and n = 7 Tacr2-/- (DSS). c, Comparison of body weight changes from baseline (day 0) to day 7, derived from the data shown in panel (b). d,e, Lipocalin-2 (LCN2) concentrations in blood (d) and feces (e) from female control and Tacr2-/- mice after 7 days of DSS or vehicle treatment, as determined by ELISA. n = 9 control (H2O), n = 7 control (DSS), n = 9 Tacr2-/- (H2O), and n = 7 Tacr2-/- (DSS) for (d); n = 6 control (H2O), n = 7 control (DSS), n = 9 Tacr2-/-(H2O), and n = 7 Tacr2-/- (DSS) for (e). f, Colon tissues from female control and Tacr2-/- mice treated with DSS or vehicle for 7 days were collected, sectioned, and stained with hematoxylin and eosin (H&E). Representative images of stained colon sections are shown. g, Representative histological images (f) were evaluated and scored according to the degree of tissue damage. n = 6 control (H2O), n = 7 control (DSS), n = 7 Tacr2-/- (H2O), and n = 6 Tacr2-/-(DSS). h, Gut permeability in female control and Tacr2-/- mice following 7 days of DSS or vehicle treatment was assessed by oral gavage of 4 kDa FITC-dextran. Blood was collected 4 hours post-gavage, and FITC fluorescence was measured in plasma to quantify gut barrier integrity. n = 9 control (H2O), n = 6 control (DSS), n = 8 Tacr2-/-(H2O), and n = 6 Tacr2-/- (DSS).

Male Tacr2-/- mice exhibit comparable gross phenotypes and feeding behaviors to control mice during chronic high-fat diet (HFD) feeding.

a, Gastrointestinal transit time was measured in male and female control and Tacr2-/-mice on a low-fat diet (LFD). n = 11 control males, n = 13 Tacr2-/- males, n = 13 control females, and n = 12 Tacr2-/- females. b, The body weights of male control and Tacr2-/-mice (8-12 weeks old) were monitored weekly over a 10-week period while maintained on a HFD. n = 7 control and n = 9 Tacr2-/- mice. c, Glucose tolerance test (GTT) was performed with 10-week-old male control (n = 6) and Tacr2-/- (n = 5) mice. d, Gastrointestinal transit time was measured in male control (n = 11) and Tacr2-/- (n = 13) mice on a HFD. e, Fecal lipid content was quantified from stool samples collected at week 2 of HFD feeding in male control (n = 6) and Tacr2-/-(n = 5) mice. At week 10 of HFD treatment, male control and Tacr2-/-mice were placed in metabolic chambers, and food intake (f,g), locomotion (h), VO2 (i), VCO2 (j), and respiratory exchange ratio (RER, k) were analyzed. n = 5 control males (Light), n = 6 Tacr2-/- males (Light), n = 5 control males (Dark), n = 5 Tacr2-/- males (Dark) for (f,g); n = 4 control males (Light), n = 6 Tacr2-/- males (Light), n = 4 control males (Dark), n = 6 Tacr2-/- males (Dark) for (h,i,j,k). Data were analyzed using two-way ANOVA with repeated measures (b,c) or Holm-Sidak post hoc analysis (f-k), or by unpaired two-tailed Student’s t-test (a,d,e). Data are shown as mean ± SEM; nsp>0.05.

Female Tacr2-/- mice exhibit comparable gross phenotypes and feeding behaviors to control mice during chronic high-fat diet (HFD) feeding.

a, Heat maps display Z-scores of normalized expression levels for genes encoding lipid droplet-associated proteins in female mice across experimental conditions. Hierarchical clustering of individual samples (top dendrogram) and genes (left dendrogram) is shown, with branch lengths reflecting the similarity between clusters. Wild-type control (WT) samples are labeled in black text, Tacr2-/- (KO) samples in red text, and samples from mice on a high-fat diet (HFD) are indicated in bold. b, The body weights of female control and Tacr2-/- mice (8-12 weeks old) were monitored weekly over a 10-week period while maintained on a HFD. n = 8 control and n = 9 Tacr2-/-mice. c, Glucose tolerance test (GTT) was performed with 10-week-old female control (n = 5) and Tacr2-/- (n = 7) mice. d, Gastrointestinal transit time was measured in female control (n = 13) and Tacr2-/- (n = 12) mice on a HFD. e, Fecal lipid content was quantified from stool samples collected at week 2 of HFD feeding in female control (n = 6) and Tacr2-/- (n = 5) mice. At week 10 of HFD treatment, female control and Tacr2-/-mice were placed in metabolic chambers, and food intake (f,g), locomotion (h), VO2 (i), VCO2 (j), and respiratory exchange ratio (RER, k) were analyzed. n = 4 control females (Light), n = 5 Tacr2-/- females (Light), n = 4 control females (Dark), n = 5 Tacr2-/- females (Dark) for (f,g); n = 4 control females (Light), n = 6 Tacr2-/- females (Light), n = 4 control females (Dark), n = 6 Tacr2-/- females (Dark) for (h,i,j,k). Data were analyzed using two-way ANOVA with repeated measures (b,c) or Holm-Sidak post hoc analysis (f-k), or by unpaired two-tailed Student’s t-test (d,e). Data are shown as mean ± SEM; *p<0.05, nsp>0.05.

Tacr2-/-mice show similar gene expression profiles in perigonadal white adipose tissues.

a, qPCR analysis of Tacr2, Adipoq, Pparg, and Leptin mRNA expression in adipocyte and mesenchymal cell fractions isolated from wild-type mice (n = 5 biological replicates). Adipoq, Pparg, and Leptin serve as adipocyte marker genes. b, Schematic of the experimental workflow. Control and Tacr2-/-mice were fed a high-fat diet (HFD) for either 1 or 12 weeks, followed by sacrifice and tissue collection. Perigonadal white adipose tissue (WAT) was harvested, total RNA was extracted, and samples were subjected to RNA sequencing. Panel created with BioRender.com/7r4281j. c,e,g,i, Principal component analysis (PCA) of RNA-Seq data from male (c,e) and female (g,i) control and Tacr2-/- mice following HFD feeding for 1 or 12 weeks. n = 3 control (1 week HFD), n = 4 control (12 week HFD), n = 3 Tacr2-/-(1 week HFD), n = 3 Tacr2-/- (12 week HFD) for (c,e); n = 3 control (1 week HFD), n = 4 control (12 week HFD), n = 3 Tacr2-/- (1 week HFD), n = 4 Tacr2-/-(12 week HFD) for (g,i). d,f,h,j, Volcano plots of differentially expressed genes in male (d,f) or female (h,j) Tacr2-/- versus control mice under 1 or 12 weeks HFD conditions. Data were analyzed using unpaired two-tailed Student’s t-test. Data are shown as mean ± SEM; **p<0.01, *p<0.05, nsp>0.05; black circles represent control mice, and red circles represent Tacr2-/-mice.

qPCR primers used in this study.

Genotyping primers used in this study.