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. 2019 Mar 19;116(12):5564-5569.
doi: 10.1073/pnas.1812901116. Epub 2019 Feb 28.

Infection by the parasitic helminth Trichinella spiralis activates a Tas2r-mediated signaling pathway in intestinal tuft cells

Xiao-Cui Luo  1 , Zhen-Huang Chen  1 , Jian-Bo Xue  1 , Dong-Xiao Zhao  1 , Chen Lu  1 , Yi-Hong Li  1 , Song-Min Li  1 , Ya-Wen Du  1 , Qun Liu  1 , Ping Wang  2 , Mingyuan Liu  3   4 , Liquan Huang  5   6
Affiliations

Infection by the parasitic helminth Trichinella spiralis activates a Tas2r-mediated signaling pathway in intestinal tuft cells

Xiao-Cui Luo et al. Proc Natl Acad Sci U S A. .

Abstract

The parasitic helminth Trichinella spiralis, which poses a serious health risk to animals and humans, can be found worldwide. Recent findings indicate that a rare type of gut epithelial cell, tuft cells, can detect the helminth, triggering type 2 immune responses. However, the underlying molecular mechanisms remain to be fully understood. Here we show that both excretory-secretory products (E-S) and extract of T. spiralis can stimulate the release of the cytokine interleukin 25 (IL-25) from the mouse small intestinal villi and evoke calcium responses from tuft cells in the intestinal organoids, which can be blocked by a bitter-taste receptor inhibitor, allyl isothiocyanate. Heterologously expressed mouse Tas2r bitter-taste receptors, the expression of which is augmented during tuft-cell hyperplasia, can respond to the E-S and extract as well as to the bitter compound salicin whereas salicin in turn can induce IL-25 release from tuft cells. Furthermore, abolishment of the G-protein γ13 subunit, application of the inhibitors for G-protein αo/i, Gβγ subunits, and phospholipase Cβ2 dramatically reduces the IL-25 release. Finally, tuft cells are found to utilize the inositol triphosphate receptor type 2 (Ip3r2) to regulate cytosolic calcium and thus Trpm5 activity, while potentiation of Trpm5 by a sweet-tasting compound, stevioside, enhances tuft cell IL-25 release and hyperplasia in vivo. Taken together, T. spiralis infection activates a signaling pathway in intestinal tuft cells similar to that of taste-bud cells, but with some key differences, to initiate type 2 immunity.

Keywords: Gαo; Gβ1γ13; Ip3r2; type 2 immunity; α-gustducin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Tas2r receptors sense and initiate the response to the helminth T. spiralis. (A) Ts extract of muscle larvae (Ts ext.) stimulated the small intestinal villi to release significantly more IL-25 than the vehicle treatment; preincubation with AITC (Ts ext.+AITC) significantly reduced IL-25 release (n = 4). Representative traces of Ca2+ responses to Ts ext. (B) and to Ts E–S (C) are shown from tuft cells of small intestinal organoids derived from a Trpm5-lacZ heterozygous mouse. Red fluorescence was used to identify tuft cells (Insets). (D) qPCR showed that Ts infection increased expression of eight Tas2rs—Tas2r108, Tas2r110, Tas2r114, Tas2r117, Tas2r122, Tas2r130, Tas2r136, and Tas2r143—and down-regulated eight others in the small intestinal villi. (E) qPCR showed that IL-13 treatment increased expression of 3 Tas2rs—Tas2r117, Tas2r136, and Tas2r143—and down-regulated 11 others in the intestinal organoids. (F) Fluorescent images of the Ca2+-sensitive dye Fluo-4 AM-loaded HEK293 cells cotransfected with Tas2r143/Gα16-gust44 with a cotransfection efficiency of 80%, before (HBSS) and after Ts ext. stimulation (Ts ext.). About 28% of the Fluo-4 AM-loaded cells responded to Ts ext. Three representative responding cells are marked by arrowheads. (G) Traces of Ca2+ responses from the three marked cells in F. (H) Salicin stimulation released significantly more IL-25 than the vehicle treatment from the small intestinal villi; preincubation with AITC (Salicin+AITC) significantly reduced IL-25 release. *P < 0.05; **P < 0.01; ***P < 0.001. (Scale bars, 50 μm.)
Fig. 2.
Fig. 2.
Intestinal tuft cells highly express Gαo, α-gustducin, Gβ1, and Gγ13. (A) Ts infection altered G-protein subunit expression in the small intestinal villi. qPCR analyses show that Ts infection for 2 wk significantly increased the expression of Gnao1-B, Gnat3, Gna15, Gnb5, Gng7, and Gng13, but decreased 13 others. (B) IL-13 treatment of the intestinal organoids up-regulated the expression of Gnao1-B, Gnat3, Gna15, Gnb1, Gnb5, and Gng13 but decreased 6 others. *P < 0.05; **P < 0.01; ***P < 0.001. (C–H) Images of double immunostaining show that Dclk1 (green) is overlapped with α-gustducin, Gαo, Gβ1, and Gγ13 (arrowheads in C, D, F, and H) but not with Gα15 or Gβ5 (arrows in E and G). Note: one epithelial cell expressed Gαo but not Dclk1 (D, arrow) while Gβ1 was enriched at the tips of tuft cells (F, arrowhead). (Scale bar, 20 μm.)
Fig. 3.
Fig. 3.
G-protein inhibitors and Gγ13 knockout abolish tuft-cell response to Ts extract. Ts ext. evoked the release of significantly more IL-25 from the intestinal villi compared with vehicle control. Preincubation with the Gαi/o inhibitor pertussis toxin (PT) (A), Gαo peptide inhibitor (Go inhib) (B), or Gβγ inhibitor gallein (C) significantly reduced Ts ext.-induced IL-25 release. (D) Tamoxifen-induced Gng13 knockout in the Lgr5-EGFP-IRES-CreERT2:Gng13flox/flox mice significantly reduced Ts ext.-induced IL-25 release compared with WT control. *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 4.
Fig. 4.
Tuft cells utilize the Plcβ2-Ip3r2-Trpm5–signaling pathway to transduce parasitic signals. (A) Double immunostaining on intestinal sections shows the colocalization of Plcβ2 (red) with Dclk1 (green) to tuft cells. (B) Ts ext. significantly increased the release of IL-25 from the intestinal villi compared with the vehicle control whereas preincubation with the phospholipase C inhibitor U73122 significantly reduced the IL-25 release. (C and D) Double immunostaining shows that Ip3r2 (C, red), but not Ip3r3 (D, red), is coexpressed with Dclk1 (green) in intestinal tuft cells. Instead, Ip3r3 is expressed in the lamina propria. (Scale bar, 20 μm.) (E and F) ELISAs show that both Ts ext. and 124 μM stevioside stimulated WT but not Trpm5−/− duodenal villi to release IL-25. (G) Both 5 mM succinate and 0.5 mM stevioside, but not water in the mock treatment, significantly increased tuft-cell abundance in the duodenums of Trpm5-lacZ+/− mice. **P < 0.01; ***P < 0.001.
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