MRGPRX4 is a G protein-coupled receptor activated by bile acids that may contribute to cholestatic pruritus

Abstract

Patients suffering from cholestasis, the slowing or stoppage of bile flow, commonly report experiencing an intense, chronic itch. Numerous pruritogens are up-regulated in cholestatic patient sera, including bile acids (BAs). Acute injection of BAs results in itch in both mice and humans, and BA-modulating therapy is effective in controlling patient itch. Here, we present evidence that human sensory neuron-expressed Mas-related G protein-coupled receptor X4 (MRGPRX4), an orphan member of the Mrgpr family of GPCRs, is a BA receptor. Using Ca²⁺ imaging, we determined that pathophysiologically relevant levels of numerous BAs activated MRGPRX4. No mouse Mrgpr orthologs were activated by BAs. To assess the in vivo relevance of BA activation of MRGPRX4, we generated a humanized mouse with targeted expression of MRGPRX4 in itch-encoding sensory neurons. BAs activated MRGPRX4⁺ sensory neurons at higher levels compared with WT neurons. Compared with control animals, MRGPRX4⁺ mice scratched more upon acute injection of BAs and in a model of cholestatic itch. Overall, these data suggest that targeting MRGPRX4 is a promising strategy for alleviating cholestatic itch.

Keywords: MRGPRX4; bile acids; cholestasis; itch; pruritus.

Fig. 1.

BAs activate MRGPRX4, a human sensory neuron-expressed GPCR. (A) The molecular stereostructures of primary and secondary BAs and their metabolic precursor, cholesterol. Major structural differences are highlighted in red. R1 denotes the potential presence of an α-hydroxyl substituent in some BA derivatives. R2 denotes additional substituents that vary among BAs. (B–H) Ca²⁺ imaging of HEK293 cells stably expressing MRGPRX4. (B) Representative Fura-2 fluorescence heat map images of HEK cells showing changes in intracellular [Ca²⁺] induced by DCA (10 µM). (Scale bar, 20 µm.) (C and F) Concentration–Ca²⁺ response curves of BAs against MRGPRX4. Data are a representative experiment of three independent replicates performed in triplicate, depicted as mean ± SEM (C) Concentration–Ca²⁺ response curves of unconjugated BAs. OA, oleic acid. (F) Concentration–Ca²⁺ response curves of tauro-conjugated BAs. (D, E, G, and H) Ca²⁺ imaging traces. A total of 10 µM DCA was added where indicated by black bars. Mean ± 95% CI depicted. n = 15. (E, G, and H) Cells were preincubated with either 10 µM sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (E), 10 µM of the G_αq inhibitor YM254890 (G), or 10 µM PLC inhibitor U73122 (H) for 30 min before imaging.

Fig. 2.

The +X4 humanized mouse sensory neurons are more readily activated by BAs. (A) Diagram showing mating strategy to generate humanized (+X4) mice. Mrgpra3-Cre expression is restricted to mouse DRG. Approximately 5% of DRG would be expected to express receptor. qPCR (B and C) RT-PCR of RNA collected from both control (A3-Cre negative) and +X4 (A3-Cre positive) mice expressing Rosa26^lsl-MRGPRX4. (B) Mean ± 95% CI depicted. n = 3 representative samples. ***P < 0.001, Student’s t test. (C) Arrow indicates the expected band based on primer design. Four independent samples collected from different mice are depicted. (D and H) Ca²⁺ imaging traces of Ctrl and +X4 DRG neurons. Mean ± 95% CI depicted. n = 10 neurons. After a 10-s baseline, either 10 µM DCA (D) or 100 µM UDCA (H) was applied where indicated by black bars. After 1 min, 1 mM CQ was added. (E and I) Percent activation of Ctrl and +X4 DRG neurons from either 10 µM DCA (E) or 100 µM UDCA (I). (F and J) Percent overlap of CQ-activated Ctrl and +X4 DRG neurons (∼5% of total) activated by both DCA and CQ (F) and both UDCA and CQ (J). (E, F, I, and J) *P < 0.05; **P < 0.01; χ² test. A neuron was considered to be activated if ∆F > 0.2 for at least 30 s. (G and K) Venn diagrams of DCA- (G), UDCA- (K), and CQ-activated neurons as a percent of total DRG neurons.

Fig. 3.

Mice expressing MRGPRX4 scratch more after injection with unconjugated and conjugated BAs. (A) Scratching bouts associated with injection of DCA. A total of 50 µL of 1 mM DCA was injected into the nape of Ctrl and +X4 mice. Ctrl n = 13; +X4 n = 6. (B) Scratching bouts associated with injection of TDCA. A total of 50 µL of 1 mM TDCA was injected into the nape of Ctrl and +X4 mice. Ctrl n = 9; +X4 n = 5. (C) Scratching bouts associated with injection of UDCA. A total of 50 µL of 2 mM UDCA was injected into the nape of Ctrl and +X4 mice. Ctrl n = 6; +X4 n = 6. (D) Scratching bouts associated with injection of CDCA. A total of 50 µL of 2 mM CDCA was injected into the nape of Ctrl and +X4 mice. Ctrl n = 6; +X4 n = 6. (E) Scratching bouts associated with injection of human plasma from either normal or cholestatic patients (Chol) into Ctrl and +X4 mice. A total of 50 µL of plasma was injected into the nape of mice. For normal plasma: Ctrl n = 5; +X4 n = 7. For Chol plasma: Ctrl n = 5; +X4 n = 5. (A–D) Mean ± SEM depicted. Each open circle represents an individual mouse. (A–D) *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed unpaired Student’s t test.

Fig. 4.

The +X4 humanized mice display increased cholestatic itch concordant with serum BA elevation. (A) Experimental flowchart of cholestatic model. ANIT was given daily per os for 5 d. After 24-h treatment, spontaneous itch was assessed for 1 h. (B) Spontaneous scratching bouts from ANIT-treated animals. Itch was assessed for 1 h. Scratching bouts from Ctrl and +X4 were significantly different. P < 0.05 by ANOVA. For day 0: Ctrl n = 5, +X4 n = 7; for day 1: Ctrl n = 14, +X4 n = 11; for day 2: Ctrl n = 13, +X4 n = 9; for day 3: Ctrl n = 11, +X4 n = 9; for day 4: Ctrl n = 10, +X4 n = 9; and for day 5: Ctrl n = 7, +X4 n = 9. (C) Percent weight change from baseline of Ctrl and +X4 mice during ANIT treatment. For day 1: Ctrl n = 5, +X4 n = 7; for all other days: Ctrl n = 4, +X4 n = 6. (D) Plasma BA levels from Ctrl and +X4 mice at indicated days of ANIT treatment. For day 0: Ctrl n = 4, +X4 n = 4; for day 1: Ctrl n = 6, +X4 n = 9; for day 5: Ctrl n = 6, +X4 n = 7. (E) Total plasma bilirubin levels at indicated days of treatment. For day 1: Ctrl n = 3, +X4 n = 3; for day 5: Ctrl n = 5, +X4 n = 9. (B–E) Mean ± SEM depicted. Circles represent individual mice. n.s., not significant; *P < 0.05; **P < 0.01; Student’s t test.