Significance: Cholestatic itch is a debilitating symptom that affects hundreds of thousands of patients every year, drastically decreasing patient quality of life. Additional therapeutic targets are needed, as current therapies are ineffective for many patients. In recent years, the Mas-related G protein-coupled receptor (MRGPR) family of receptors has been linked to itch. Here, we determine that MRGPRX4 is specifically activated by numerous bile acids (BAs) up-regulated in cholestatic patient sera. To test the relevance of these findings in vivo, we generated humanized mice expressing MRGPRX4 in sensory neurons. MRGPRX4 ⁺ humanized mice display higher levels of itch in response to injection of BAs and in a mouse model of cholestasis, indicating that MRGPRX4 could play a role in cholestatic itch.

Various pathologic conditions result in jaundice, a yellowing of the skin due to a buildup of bilirubin. Patients with jaundice commonly report experiencing an intense non-histaminergic itch. Despite this association, the pruritogenic capacity of bilirubin itself has not been described, and no bilirubin receptor has been identified. Here, we demonstrate that pathophysiologic levels of bilirubin excite peripheral itch sensory neurons and elicit pruritus through MRGPRs, a family of G-protein coupled receptors expressed in primary sensory neurons. Bilirubin binds and activates two MRGPRs, mouse MRGPRA1 and human MRGPRX4. In two mouse models of pathologic hyperbilirubinemia, we show that genetic deletion of either Mrgpra1 or Blvra, the gene that encodes the bilirubin-producing enzyme biliverdin reductase, attenuates itch. Similarly, plasma isolated from hyperbilirubinemic patients evoked itch in wild-type animals but not Mrgpra1^-/- animals. Removing bilirubin decreased the pruritogenic capacity of patient plasma. Based on these data, targeting MRGPRs is a promising strategy for alleviating jaundice-associated itch.

Reflexes initiated by the carotid body, the principal O₂-sensing organ, are critical for maintaining cardiorespiratory homeostasis during hypoxia. O₂ sensing by the carotid body requires carbon monoxide (CO) generation by heme oxygenase-2 (HO-2) and hydrogen sulfide (H₂S) synthesis by cystathionine-γ-lyase (CSE). We report that O₂ stimulated the generation of CO, but not that of H₂S, and required two cysteine residues in the heme regulatory motif (Cys²⁶⁵ and Cys²⁸²) of HO-2. CO stimulated protein kinase G (PKG)–dependent phosphorylation of Ser³⁷⁷ of CSE, inhibiting the production of H₂S. Hypoxia decreased the inhibition of CSE by reducing CO generation resulting in increased H₂S, which stimulated carotid body neural activity. In carotid bodies from mice lacking HO-2, compensatory increased abundance of nNOS (neuronal nitric oxide synthase) mediated O₂ sensing through PKG-dependent regulation of H₂S by nitric oxide. These results provide a mechanism for how three gases work in concert in the carotid body to regulate breathing.