Fig 2: MPECs maintain AhR activity via IL-2. (A and B) C57BL/6 mice (CD45.2+) were transferred with 1 × 105 CD45.1+ CD8+ OT-I T cells, followed by infection of Lm-OVA. (A) AhR expression in IL-2high and IL-2low CD8+ OT-I Teff cells was analyzed 7 d after infection (n = 3). (B) mRNA levels of Cyp1a1 and Cyp1b1 in IL-2high and IL-2low CD8+ T cells (n = 3). (C) Percentage of IL-2-producing OT-I T cells in MPEC and SLEC (n = 6 mice per group). (D) 5HTP abundance in MPEC and SLEC was analyzed by LC/MS (n = 3). (E and F) C57BL/6 mice (CD45.2+) were transferred with 1 × 105 CD45.1+CD45.2+CD8+ OT-I T cells, followed by infection of Lm-OVA 24 h later. Mice were then treated with STAT5-IN-1 (STAT5 inhibitor) or CP-10188 (Tph1 inhibitor). (E) Proportions of MPEC and SLEC were measured at day 10 post Lm-OVA infection (n = 5 mice per group). (F) mRNA levels of Cyp1a1 and Cyp1b1 in MPEC were analyzed by qPCR. (G and H) MPEC were purified from 10-d infected mice, followed by IL-2 neutralizing antibody treatment for 24 h in vitro (n = 3). (G) 5HTP abundance was measured by LC/MS. (H) mRNA levels of Cyp1a1 and Cyp1b1 were analyzed. Data are representative of ≥3 independent experiments (mean ± SD). P values were calculated using unpaired two-tailed Student’s t test (A–D, G, and H) and or one-way ANOVA (E and F). *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 3: Tph1 KO mice constructed using Tph1tm1Kry retains partial TPH activity. (a) Genomic structure of Tph1tm1Kry. (b) Concentration of jejunal 5-HT in wild type and Tph1tm1Kry GKO mice (n = 3–7 per group). All data are presented as means ± standard error (**P < 0.01 vs. wild type, Student’s t-test). (c) Representative immunohistochemical staining of 5-HT in the jejunum of wild type and Tph1tm1Kry GKO mice. Scale bar, 50 μm. (d) Representative immunofluorescence staining of pancreatic islets in Tph1tm1Kry βKO mice (n = 3 per group). Pancreatic sections were stained for insulin (green) and 5-HT (red). Control: MIP-Cre-hGH; βKO: Tph1tm1Kry βKO. Scale bar, 50 μm. (e) The amino acid sequence of TPH1. Red letters indicate overlapping splice sites; RD, regulatory domain; CD, catalytic domain; TD, tetramerization domain. (f) Tph1 transcripts of wild type mice and KO transcripts of Tph1tm1Kry GKO mice in the duodenum were amplified by RT-PCR using the primers, Tph1 Exon1 forward and Tph1 Exon11 reverse. Empty triangles indicate 1000 bp, and black triangles indicate 1500 bp. The full-length gel is presented in Supplementary Figure S3. (g) TPH1 (50 kD) and truncated TPH1 (37 kD) were detected by immunoblot analyses of pancreatic islets from Tph1tm1Kry βKO and hetero βKO mice. The full-length blot is presented in Supplementary Figure S2.

Fig 4: Tph1 expression and 5-HT levels in the Tph1tm1a mouse. (a) Tph1 mRNA levels in the jejunum of Tph1tm1a mice were determined by quantitative real time RT-PCR and expressed relative to those of wild type mice. (b) Representative immunohistochemical staining of 5-HT in the jejunum of wild type and Tph1tm1a mice. Scale bar, 50 μm. (c) Levels of 5-HT in serum, the jejunum, and brain were determined by LC-MS/MS. All data are presented as means ± standard error (n = 5–8 per group; *P < 0.05, **P < 0.01, and ***P < 0.001 vs. Tph1+/+, Student’s t-test).

Fig 5: Effects of different phytochemicals and Trp metabolites on the gene expression of 5-HT metabolism-related enzymes, including Tph1 (A) and Maoa (B) in the ileum of mice. The data in the charts are the means ± SEMs. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to the control group. Tph1, tryptophan hydroxylase 1; Maoa, monoamine oxidase A; VMA, vanillylmandelic acid; PA, perillic acid; MurA, muramic acid; CA, cinnamic acid; CGA, chlorogenic acid; NAS, N-acetylserotonin; ILA, indolelactic acid; IAld, indoleacetaldehyde. The number of animals is shown in Table 1.