Extended Data Fig. 6: Effects of the tryptophan (Trp) diet and metabolites I3A, IPyA, and IEt in protecting against C. rodentium infection depend on dopamine receptor D2 (DRD2) in intestinal epithelial cells (IECs).
From: Dopamine receptor D2 confers colonization resistance via microbial metabolites
Drd2fl/fl x Villin (Vil)-Cre or Drd2fl/fl mice were fed a conventional (2 g Trp/kg diet, ad libitum) or Trp (42 g Trp/kg diet, ad libitum) diet for 7 d or Trp metabolites, I3A (1000 mg/kg), IPyA (2900 mg/kg), or IEt (600 mg/kg), by oral gavage daily for 2 d, and then infected with C. rodentium (CR, oral gavage, 108 CFU) with continued Trp feeding or metabolite treatment. a–b, Bacterial load in (a) feces and (b) colon tissue was measured (a) every 1–2 d for 24 d post-infection and (b) at the peak of infection, 10 d post-infection. c–e, Colon sections were stained with H&E and (c) blindly scored for submucosal edema (0-3), goblet cell depletion (0-3), epithelial hyperplasia (0-3), epithelial integrity (0-4), and neutrophil and mononuclear cell infiltration (0-3). Data are expressed as the sum of these individual scores (0-16). See Methods for full description of scoring rubric. (d) Crypt heights were measured. (e) Representative images. Scale bar: 50 μm. (f) Representative images of pedestals from Fig. 4a (denoted by arrows) stained with DAPI and Alexa Fluor 647-phalloidin and imaged by confocal microscopy. Shown are maximum intensity z-projections. Scale bar: 5 μm. (g) Pedestal formation = # of pedestals per host cell (I3A, n = 1079; IPyA, n = 1027 cells examined over 3 independent experiments). For box plots, interquartile ranges (IQRs, boxes), median values (line within box), whiskers (lowest and highest values within 1.5 times IQR from the first and third quartiles), and outliers beyond whiskers (dots), are shown. h, i, Intestinal epithelial cells were isolated, and cell lysates were analyzed by Western blotting with the indicated antibodies. Source data are provided in Supplementary Fig. 9. (i) Densitometry was performed using FIJI, n = 3 biological replicates. Data are representative of at least 3 independent experiments, n = 10 mice per group, bars = mean, error bars = standard deviation. Statistical analysis was performed using the two-tailed Student’s t-test (a) or one-way ANOVA, followed by post-hoc Tukey multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001.
