Interleukin-33 Promotes Serotonin Release from Enterochromaffin Cells for Intestinal Homeostasis

Abstract

The gastrointestinal tract is known as the largest endocrine organ that encounters and integrates various immune stimulations and neuronal responses due to constant environmental challenges. Enterochromaffin (EC) cells, which function as chemosensors on the gut epithelium, are known to translate environmental cues into serotonin (5-HT) production, contributing to intestinal physiology. However, how immune signals participate in gut sensation and neuroendocrine response remains unclear. Interleukin-33 (IL-33) acts as an alarmin cytokine by alerting the system of potential environmental stresses. We here demonstrate that IL-33 induced instantaneous peristaltic movement and facilitated Trichuris muris expulsion. We found that IL-33 could be sensed by EC cells, inducing release of 5-HT. IL-33-mediated 5-HT release activated enteric neurons, subsequently promoting gut motility. Mechanistically, IL-33 triggered calcium influx via a non-canonical signaling pathway specifically in EC cells to induce 5-HT secretion. Our data establish an immune-neuroendocrine axis in calibrating rapid 5-HT release for intestinal homeostasis.

Keywords: IL-33; PLC-γ1; ST2; TRPA1; enterochromaffin cells; gut motility; helminth clearance; serotonin release.

Figure 1.. IL-33 promotes parasite expulsion in a type 2 immune response-independent manner.

(A-E) T. muris eggs were inoculated into WT and Il4^−/−Il13^−/− mice by oral gavage. The mice were treated with PBS or IL-33 daily from day 7 and sacrificed on 14 day for further test. (A) IL-4, IL-5 and IL-13 protein concentrations in mesenteric lymph nodes (mLN) were assessed by ELISA. (B) PAS staining and (C) Quantification of goblet cells per crypt of colonic tissues. Scale bar, 50 μm. (D) Quantification of larvae in the colons of WT and Il4^−/−Il13^−/− mice. (E) T. muris infected WT and Il4^−/−Il13^−/− mice were treated daily with PBS or IL-33 for 7 days. Colonic transit time was assessed by bead expulsion assay. Data are representative of three independent experiments (A-E). NS, not significant; ND, not detected; *p < 0.05 (Student’s t-test, error bars represent SD). Please also see Figure S1.

Figure 2.. IL-33-ST2 signal regulates gut motility.

(A) WT mice were treated daily with IL-33 or PBS for 7 days. Colonic transit time was assessed by bead expulsion assay. (B) Colonic transit time was measured by bead expulsion assay in WT, Il33^−/− and Il1rl1^−/− mice. (C) Representative trace and (D) Quantification of colon contraction in WT, Il33^−/− and Il1rl1^−/− mice. (E) WT, Il33^−/− and Il1rl1^−/− mice were treated daily with PBS or IL-33 for 7 days. Colonic transit time was assessed by bead expulsion assay at day 8. (F) WT, Il33^−/− and Il1rl1^−/− mice were treated with PBS or IL-33, and waited for 10 minutes. Colonic transit time measured by bead expulsion assay. (G) Representative trace of IL-33-induced colon contraction in WT, Il33^−/− and Il1rl1^−/− mice. Data are representative of three independent experiments (C, G) or are pooled from two independent experiments (A, B, D-F). NS, not significant; *p < 0.05 (Student’s t-test, error bars represent SD). Please also see Figure S2.

Figure 3.. IEC-derived ST2 is required for gut motility.

(A-C) Colonic transit time was assessed by bead expulsion assay in the indicated mouse strains. (D) Il1rl1^fl/fl and Vil1^creIl1rl1^fl/fl mice were treated with PBS or IL-33, and waited for 10 minutes. Colonic transit time was assessed by bead expulsion assay. (E) Representative trace of IL-33-induced colon contraction in Il1rl1^fl/fl and Vil1^creIl1rl1^fl/fl mice. (F) GCaMP3 fluorescence signal of neurons on the myenteric plexus of Pirt^GCaMP3 mice treated with IL-33 and mCPBG (5HT₃R agonist). (G) Time-resolved responses (ΔF/F, color scale) of neurons (one neuron per row) and (H) Quantification of IL-33 responding neurons in the myenteric plexus with or without epithelium. mCPBG was used as a positive control. Data are representative of three independent experiments (E-G) or are pooled from two independent experiments (A-D, H). NS, not significant; *p < 0.05; **p < 0.01 (Student’s t-test, error bars represent SD). Please also see Figure S3.

Figure 4.. IL-33 induces 5-HT secretion for gut motility.

(A) WT mice were i.p. injected with PBS or IL-33 and waited for 10 minutes. The IEC-derived secretome was assessed from the mice serum. (B) WT mice were treated with IL-33. Mice serum was collected at the indicated time point, and relative 5-HT amounts were assessed by ELISA. (C) Tph1^fl/fl and Vil1^creTph1^fl/fl mice were treated with PBS or IL-33, and waited for 10 minutes. Mice serum was collected, and relative 5-HT amounts were assessed by ELISA. (D) Time-resolved responses (ΔF/F, color scale) of neurons (one neuron per row) and (E) Quantification of IL-33 responding neurons in the myenteric plexus treated with the indicated conditions. K⁺ was used as a positive control. (F) Tph1^fl/fl and Vil1^creTph1^fl/fl mice were treated with PBS or IL-33, and waited for 10 minutes. Colonic transit time was measured by bead expulsion assay. (G) Representative trace of IL-33-induced colon contraction in Tph1^fl/fl and Vil1^creTph1^fl/fl mice. Data are representative of three independent experiments (A-D, G) or are pooled from two independent experiments (E, F). NS, not significant; *p < 0.05; **p < 0.01 (Student’s t-test, error bars represent SD). Please also see Figure S4.

Figure 5.. EC cell-derived ST2 responds to IL-33 for 5-HT release.

(A) Representative immunofluorescence staining for 5-HT, chromogranin A (ChgA), ST2 with DAPI in the intestinal tissues of WT mice. Scale bar, 10 μm. (B) Il1rl1^fl/fl and Chga^creERIl1rl1^fl/fl mice were treated with PBS or IL-33, and waited for 10 minutes. Mice serum was collected, and relative 5-HT amounts were assessed by ELISA (C) Time-resolved responses (ΔF/F, color scale) of neurons (one neuron per row) and (D) Quantification of IL-33 responding neurons in the myenteric plexus. mCPBG (5HT₃R agonist) was used as a positive control. (E) Il1rl1^fl/fl and Chga^creERIl1rl1^fl/fl mice were treated with PBS or IL-33, and waited for 10 minutes. Colonic transit time was measured by bead expulsion assay. (F) Representative trace of IL-33-induced colon contraction in Il1rl1^fl/fl and Chga^creERIl1rl1^fl/fl mice. (G-J) T. muris eggs were inoculated into Il1rl1^fl/fl and Chga^creERIl1rl1^fl/fl mice (pre-treated with tamoxifen) by oral gavage and the mice were sacrificed on day 14 for further testing. (G) IL-4, IL-5 and IL-13 protein concentrations from mLN were assessed by ELISA. (H) PAS staining and (I) Quantification of goblet cells per crypt in colonic tissues. Scale bar, 50 μm. (J) Quantification of larvae in the colonic tissues. Data are representative of two independent experiments (A-C, F-J) or are pooled from two independent experiments (D, E). NS, not significant; *p < 0.05; **p < 0.01 (Student’s t-test, error bars represent SD). Please also see Figure S5.

Figure 6.. TRPA1 is required for IL-33-mediated 5-HT release.

(A) Representative time-lapse Ca²⁺ imaging traces of IL-33-induced [Ca²⁺]_i response in NIH 3T3 cells overexpressed with ST2 alone; or with TRPA1 alone; or with both ST2 and TRPA1. Ionomycin and AITC were used as positive controls. (B) Representative trace and (C) Quantification of IL-33-induced action potentials were recorded by whole-cell patch-clamp from isolated EC cells of Tph1^CFP mice with or without A967079 (TRPA1 antagonist), or isolated EC cells of Trpa1^−/−Tph1^CFP mice. K⁺ was used as a positive control. Inset: representative action potential. Scale bar, 20 mV, 10 ms. (D) Relative 5-HT amounts in the cultured supernatant from EC cell-enriched intestinal organoids treated with PBS, IL-33 or AITC (TRPA1 agonist) for 8 hours were assessed by ELISA. (E) Relative 5-HT amounts in the cultured supernatant from EC cell-enriched intestinal organoids treated with the indicated conditions for 8 hours derived from Tph1^CFP mice were assessed by ELISA. A967079 (TRPA1 antagonist). ω-agatoxin IVA (Ca²⁺ channel blocker). (F) Representative immunofluorescence staining for 5-HT, ST2, TRPA1 and DAPI in the colonic tissues of WT mice. Scale bar, 10 μm. (G) Trpa1^fl/fl and Chga^creERTrpa1^fl/fl mice were treated with PBS or IL-33, and waited for 10 minutes. Mice serum was collected, and relative 5-HT amounts were assessed by ELISA. (H) Time-resolved responses (ΔF/F, color scale) of neurons (one neuron per row) and (I) Quantification of Trpa1^fl/flPrit^GCaMP3 and Chga^creERTrpa1^fl/flPrit^GCaMP3 mice that were treated with the indicated conditions. mCPBG (5HT₃R agonist) was used as a positive control. (J) Trpa1^fl/fl and Chga^creERTrpa1^fl/fl mice were treated with PBS or IL-33, and waited for 10 minutes. Colonic transit time was measured by bead expulsion assay. (K) Representative trace of IL-33-induced colon contraction in Trpa1^fl/fl and Chga^creERTrpa1^fl/fl mice. Data are representative of three independent experiments (A, B, D-H, J, K) or are pooled from two independent experiments (C, I). NS, not significant; *p < 0.05; **p < 0.01 (Student’s t-test, error bars represent SD). Please also see Figure S6.

Figure 7.. IL-33 induces PLC-γ1 activation for 5-HT release in EC cells.

(A) Representative trace and (B) Quantification IL-33-induced action potentials were recorded by whole-cell patch-clamp from isolated EC cells of Tph1^CFP mice with or without BAPTA. K⁺ was used as a positive control. Inset: representative action potential. Scale bar, 20 mV, 10 ms. BAPTA (calcium chelator, cell-impermeant). (C) Tph1^CFP EC-enriched intestinal organoids were treated with PBS or IL-33 for 10 minutes and fixed. Representative immunofluorescence staining for TPH1-CFP, p-PLC-γ1 and DAPI. Scale bar, 100 μm. (D) Representative trace and (E) Quantification of IL-33-induced action potentials were recorded by whole-cell patch-clamp in isolated EC cells from Tph1^CFP mice treated with U-73343 (inactive analog of PLC-γ1 inhibitor U-73122) or U-73122. Inset: representative action potential; K⁺ was used as a positive control. Scale bar, 20 mV, 10 ms. (F) Relative 5-HT amounts in the cultured supernatant from EC-enriched intestinal organoids treated with the indicated conditions for 8 hours derived from Tph1^CFP mice were assessed by ELISA. BAPTA-AM (calcium chelator, cell-permeant). (G) Myd88^−/−Tph1^CFP EC-enriched intestinal organoids were treated with PBS or IL-33 for 10 minutes and fixed. Representative immunofluorescence staining for TPH1-CFP, p-PLC-γ1 and DAPI. Scale bar, 100 μm. (H) Relative 5-HT amounts in the cultured supernatant from EC-enriched intestinal organoids derived from Tph1^CFP and Myd88^−/−Tph1^CFP mice treated with PBS or IL-33 for 8 hours were assessed by ELISA. (I) Representative immunofluorescence staining for 5-HT, ST2, TRPA1 and DAPI in the healthy human intestinal tissues. Scale bar, 10 μm. (J) Human EC cell-enriched intestinal organoids were treated with PBS or IL-33 for 10 minutes and fixed. Representative immunofluorescence staining for 5-HT, p-PLC-γ1 with DAPI. Scale bar, 100 μm. (K-L) The human EC cell-enriched intestinal organoids were treated with the indicated conditions for 8 hours. Relative 5-HT amounts in the cultured supernatant were assessed by ELISA. Data are representative of three independent experiments (A, C, D, F-L) or are pooled from two independent experiments (B, E). NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t-test, error bars represent SD). Please also see Figure S7.