Fig 1: The type 2 immune response induced by parasitic infections is impaired in p53-/- mice.a Schematic illustration of the signaling pathway utilized by tuft cells to initiate the type 2 immunity towards parasitic infections. b–k p53+/+ and p53-/- mice were infected with Tritrichomonas muris (Tm) by oral gavage (P.O.) and examined at 21 d.p.i. b–f or with Nippostrongylus brasiliensis (Nb) by subcutaneous (s.c.) injection and examined at 5 d.p.i. for flow cytometric analysis and 7 d.p.i. for other assays g–k. b and g Tuft cell lineage expansion was assessed by Dclk1 IHC staining in the small intestine of naïve and infected mice. Left: representative images. Right: quantifications of tuft cell number in the villi. c and h Goblet cell hyperplasia was assessed by Alcian blue staining in the small intestine of naïve and infected mice. Left: representative images. Right: quantifications of the goblet area. d and i Flow cytometric analysis of the population of eosinophils and ILC2s in the lamina propria (LP). Flow cytometry gating strategy and representative images are shown in Fig S11. e and j Relative mRNA levels of IL-13 in the small intestine of naïve and infected mice as determined by quantitative real-time PCR and normalized with ß-actin. f Tm numbers in cecum at 21 d.p.i. in p53+/+ and p53-/- mice. k Egg counts in feces and worm burden in the intestine at 7 d.p.i. in p53+/+ and p53-/- mice. For b–e, g–j, the ileum, and duodenum tissues were collected from Tm-infected and Nb-infected mice, respectively, for analysis. b–k Data are presented as mean ± SEM. Each dot represents an individual mouse. n = 5–7/group. For b and g =30 villus/mouse were counted. For c and h, =50 goblet cells/mouse were quantified. *p < 0.05; **p < 0.01; ***p < 0.001; NS: non-significant, two-tailed Student’s t-test.
Fig 2: Lrmp interacts with ITPR2 and regulates Ca2+ flux in cells.a and b Lrmp interacted with IP3R proteins, including ITPR2, as determined by co-IP followed by LC-MS/MS assays in MEFs. a Lrmp-interacting proteins (label with red) mapped in “Chemo-sensing pathway” in KEGG. b Counts of Lrmp and IP3Rs detected by co-IP followed by LC-MS/MS assays in MEFs. c Lrmp-ITPR2 interaction was confirmed by co-IP assays followed by Western-blot assays in MEFs transfected with vectors expressing Lrmp-Flag and ITPR2-HA, respectively. d The binding region of Lrmp for ITPR2 was determined by co-IP followed by Western-blot assays in H1299 cells transfected with vectors expressing different Lrmp-Flag fragments and ITPR2-HA, respectively. Upper panel: schematic representation of vectors expressing full-length (FL) or serial deletion mutants of Lrmp-Flag. e The Lrmp-IPTR2 interaction was examined by PLA assays in WT and Lrmp-/- small intestinal tissues. Red: PLA signals of the Lrmp-ITPR2 interaction; green: Dclk1; DAPI: nucleus. f–h Ca2+ flux in response to Ionomycin (2 µM) treatment in WT, p53-/- and Lrmp-/- MEFs with or without transfection of either FL- or ?M-Lrmp-Flag vectors. f Average traces of Ca2+ responses. g Relative Fluo-4 fluorescence obtained from Ca2+ transients at peak (48 s). h Representative fluorescence images. The Fluo-4 fluorescence intensity before Ionomycin treatment was calculated as 1. In f, each curve represents an average of at least 90 cells. Curves of each cell are shown in Fig S7. ***p < 0.001; **p < 0.01; NS: non-significant, two-tailed Student’s t-test.
Fig 3: p53 deficiency impairs the function of tuft cells to trigger type 2 immunity.a–c p53+/+ and p53-/- mice were given drinking water with or without 150 mM succinate (Sc) for 7 days before analysis. a. Quantifications of tuft (left) and goblet cells (right) in the small intestine of mice treated with or without succinate by IHC staining of Dclk1 and Alcian blue staining, respectively. b Quantifications of eosinophils and ILC2s in the LP by flow cytometric analysis. c Relative mRNA levels of IL-13 in the small intestine. In c, d, and g, mRNA levels were determined by quantitative real-time PCR and normalized with ß-actin. d Relative mRNA levels of IL-25 in the intestinal epithelium of naïve mice, mice infected with Tm or Nb, and mice treated with succinate. e–g p53+/+ and p53-/- mice were treated with rIL-25 (0.5 µg/day; i.p.) or PBS for 7 days before analysis. e Quantifications of tuft (left) and goblet cells (right) in the small intestine. f Quantifications of eosinophils and ILC2s in the LP. g Relative mRNA levels of IL-13 in the small intestine. h rIL-13 induced tuft cell expansion in p53+/+ and p53-/- intestinal organoids to a similar extent. The intestinal organoids from p53+/+ and p53-/- mice were treated with rIL-13 (10 ng/ml for 48 h) or PBS, and then tuft cells were detected by IF staining of Dclk1. Left: representative images. Right: quantifications of tuft cells. For a–h, data are presented as mean ± SEM. For a–g, each dot represents an individual mouse. n = 5–8/group. For h, each dot represents an image field. *p < 0.05; **p < 0.01; ***p < 0.001; NS: non-significant, two-tailed Student’s t-test.
Fig 4: The intestinal type 2 immune response induced by parasitic infections and succinate is impaired in Lrmp-/- mice.a–f WT and Lrmp-/- mice were infected with Tm or Nb and examined at 21 d.p.i. (for Tm) and 5 or 7 d.p.i. (for Nb), respectively. a and b Quantifications of tuft (a) and goblet (b) cells in the small intestine of mice by IHC staining of Dclk1 and Alcian blue staining, respectively. c and d Quantifications of eosinophils (c) and ILC2s (d) in the LP by flow cytometric analysis. e Relative IL-13 mRNA levels in the small intestine determined by quantitative real-time PCR assays. f Egg counts in feces (left) and worm burden (right) in the intestine in Nb-infected mice. g–i The impaired type 2 immune response induced by succinate (150 mM for 7 days) in Lmrp-/- mice. g Quantifications of tuft and goblet cells in the small intestine. h Quantifications of eosinophils and ILC-2s in the LP. i Relative IL-13 mRNA levels in the small intestine. Data are presented as mean ± SEM. Each dot represents an individual mouse. n = 5–8/group. *p < 0.05; **p < 0.01; ***p < 0.001; NS: non-significant, two-tailed Student’s t-test.
Fig 5: Lrmp is a p53 target gene highly expressed in tuft cells.a The putative p53-binding elements in the Lrmp gene predicted by the p53MH program. A consensus p53-binding element is also presented. Pu, purine; Py, pyramidine; N, any nucleotide. b p53 bound to two p53-binding elements in Lrmp determined by ChIP assays. c Ectopic expression of p53 transactivated the p53-binding elements in Lrmp determined by luciferase reporter assays in p53-/- MEFs. d p53 transcriptionally upregulated Lrmp expression. mRNA levels were determined by quantitative real-time PCR and normalized with ß-actin. e Lrmp expression in mouse intestinal epithelial cells by analyzing a scRNA-seq dataset (GSE92332). f Co-localization of Lrmp and Dclk1 in tuft cells determined by IF staining of intestinal organoids. g Lrmp mRNA and protein levels in p53+/+, p53-/- and Lrmp-/- tuft cells determined by quantitative real-time PCR and Western-blot assays, respectively. For b, c, d, and g, data are presented as mean ± SD. **p < 0.01; ***p < 0.001, two-tailed Student’s t-test. UD: undetectable.
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