Author response: Identification of a bilirubin receptor that may mediate a component of cholestatic itch

Field: Medicine (Health Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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. https://doi.org/10.7554/eLife.44116.001 eLife digest Jaundice causes the skin to yellow as a result of a build-up of a pigment called bilirubin. Normally, bilirubin is made in the liver and removed from the body in digestive fluid called bile, but people with liver or gallbladder problems may end up with too much bilirubin that accumulates in their blood and skin. One side effect of jaundice is intense and uncontrollable itching. Researchers are not sure what causes this itching, and there are few treatments that help to relieve it. At the molecular level, itching sensations occur when compounds bind to particular receptors on the surface of nerve cells. One family of receptors that can trigger itch is called the Mas-related G-protein Coupled Receptor (MRGPR). Could one of these receptors trigger jaundice-related itching? Now, Meixiong, Vasavda et al. show that bilirubin binds to and activates MRGPRs to cause itch in mice. Whereas injecting bilirubin into normal mice causes them to scratch, mice that have been genetically engineered to lack MRGPRs do not itch when their own bilirubin levels rise, or when they are injected with bilirubin or with plasma from patients who experience jaundice-related itching. Furthermore, removing bilirubin from the plasma of patients before it was injected into normal mice reduced the amount of itching that the mice felt. Overall, the results reported by Meixiong, Vasavda et al. suggest that drugs that prevent bilirubin from attaching to MRGPRs might help to alleviate jaundice-related itching. However, researchers must first verify that bilirubin interacts with MRGPRs in people to cause itch. If bilirubin causes itch in people like in mice, scientists could then evaluate existing drugs or make new ones to prevent bilirubin from attaching to the MRGPRs. https://doi.org/10.7554/eLife.44116.002 Introduction Chronic pruritus, or itch, is a complex and often debilitating symptom that accompanies a range of cutaneous and non-cutaneous diseases (Ständer et al., 2007; Yosipovitch and Bernhard, 2013a). The most widely known pruritogen is histamine, which is secreted by mast cells in the skin and activates histamine receptors on nearby sensory neurons (Bautista et al., 2014; Ikoma et al., 2006; LaMotte et al., 2014; Yosipovitch and Bernhard, 2013b). While viable treatments exist for histamine-mediated itch, most non-histaminergic conditions are more difficult to treat because the mediators are often unknown (Kremer et al., 2011). Jaundice, or yellowing of the skin, sclera, and mucosa due to abnormal accumulation of the yellow heme metabolite bilirubin, is commonly associated with chronic non-histaminergic pruritus. Jaundice often presents in patients with hepatobiliary disorders such as cholestasis, characterized by impaired bile flow. Physiologically, bilirubin is typically bound to albumin in serum and concentrates in the liver, where it is conjugated to glucuronic acid and subsequently excreted in bile. At physiologic and mildly elevated concentrations (0.2–2.7 mg/dL, 3.4–46.2 μM), bilirubin is benign. At highly elevated levels however, such as in cutaneous jaundice (>5 mg/dL, >85.5 μM bilirubin), it is associated with pruritus, a correlation first noted by physicians as early as the second century B.C.E. (Bassari and Koea, 2015). Results Despite the long-standing association between jaundice and pruritus (Talwalkar et al., 2003), bilirubin itself has not been described as a pruritogen. To determine whether bilirubin directly elicits pruritus, we subcutaneously injected bilirubin into the napes of mice. Pathophysiologic concentrations of bilirubin stimulated scratching in a dose-dependent manner at the site of injection (Figure 1A). Pre-incubating bilirubin with excess human serum albumin, which binds bilirubin with high affinity (Breaven et al., 1973; Griffiths et al., 1975; Jacobsen and Brodersen, 1983), elicited fewer scratches (Figure 1A). The behavioral profile of bilirubin-induced scratching mirrored that of two well-characterized pruritogens, histamine and chloroquine (Figure 1B). Notably, histamine and chloroquine only elicit itch when injected into mice at millimolar concentrations despite having nanomolar affinity towards their receptors. In comparison, bilirubin elicited a similar degree of itch even when injected at lower concentrations than histamine or chloroquine (Figure 1B). Since mice indiscriminately scratch at the nape if injected with substances that trigger either itch or pain, we also injected mice at the cheek. Unlike at the nape, painful sensations at the cheek evoke a distinct wiping behavior instead of scratching, whereas itchy sensations still elicit scratching (Shimada and LaMotte, 2008). Injecting bilirubin in the cheek prompted dose-dependent scratching just as it did at the nape (Figure 1—figure supplement 1A). Bilirubin elicited neither wiping nor licking, indicating that it selectively triggers itch and not pain (Figure 1—figure supplement 1B–C). Figure 1 with 1 supplement see all Download asset Open asset Bilirubin elicits non-histaminergic, Mrgpr-dependent pruritus. (A) Scratching bouts associated with injection of bilirubin. The indicated amount of bilirubin was injected into the nape of mice. The blue bar (+HSA) represents animals injected with 60 µg bilirubin pre-incubated with 1% human serum albumin. Veh n = 8; 6 µg n = 5, 18 µg n = 11, 30 µg n = 12, 60 µg n = 7,+HSA n = 12. (B) Time course of itch behavior associated with bilirubin, histamine, or chloroquine. Scratching bouts were binned according to 5 min intervals. Bilirubin n = 16, Histamine n = 13, Chloroquine n = 11. (C) 60 µg bilirubin was injected into the nape of WT and Mrgpr-cluster KO littermates. WT n = 8, Mrgpr-cluster KO n = 13. (D) 60 µg of the indicated metabolite was injected into WT and Mrgpr-cluster KO littermates. Hemin (WT n = 10, Mrgpr-cluster KO n = 6), Biliverdin (WT n = 7, Mrgpr-cluster KO n = 7), Urobilinogen (WT n = 15, Mrgpr-cluster KO n = 8), Stercobilin (WT n = 7, Mrgpr-cluster KO n = 5). (E) The pathway of heme degradation. The skeletal formula of each metabolite is depicted above its optimal 3D geometry, as calculated by a B3LYP functional and 6-31G(d) basis set. Blue and orange represent orbital parity of each metabolite’s HOMO obtained from DFT calculations. (A, C, D) Mean ±s.e.m. depicted. Each open circle represents an individual mouse. *, p < 0.05; **, p < 0.01; ***, p < 0.001; two-tailed unpaired Student’s t-test. https://doi.org/10.7554/eLife.44116.003 Figure 1—source data 1 Source data for Figure 1. https://doi.org/10.7554/eLife.44116.005 Download elife-44116-fig1-data1-v3.xlsx We injected mice with metabolites structurally similar to bilirubin to determine the specificity of bilirubin’s pruritic activity (Figure 1E). The two metabolites directly epistatic to bilirubin, hemin and biliverdin, did not induce scratching despite also being tetrapyrroles (Figure 1D). While hemin, biliverdin, and bilirubin display only minor atomic and electronic differences between them, they vary substantially in their physiochemical properties and structures (Figure 1E). To better understand these differences, we performed density functional theory (DFT) calculations (Becke, 1993; Hohenberg and Kohn, 1964; Kohn and Sham, 1965; Stephens et al., 1994) to determine the optimal geometry of each metabolite. Unlike in heme and biliverdin, bilirubin’s four pyrroles are extended and do not lie in the same plane (Figure 1E). DFT calculations revealed that urobilinogen and stercobilin, two bacterial metabolites derived from bilirubin, adopt a similar extended conformation. Both urobilinogen and stercobilin were able to stimulate scratching behavior (Figure 1D), indicating that bilirubin’s non-polar pyrroles may be important for its pruritic activity. Patients with jaundice-associated pruritus receive little benefit from antihistamines (Bergasa, 2014). Consistent with these clinical findings, the histamine receptor one blocker cetirizine (30 mg/kg, i.p.) failed to alleviate scratching behavior in mice injected with bilirubin (Figure 1—figure supplement 1D). Furthermore, bilirubin did not elicit a calcium response or induce appreciable histamine release from peritoneal mast cells (Figure 1—figure supplement 1E–F). The Mas-related G-protein coupled receptor (Mrgpr) family of receptors is a major mediator of non-histaminergic pruritus (Han et al., 2013; Liu et al., 2012; Liu et al., 2009; Sikand et al., 2011). To test whether Mrgprs mediate bilirubin-induced pruritus, we injected mice lacking a cluster of 12 Mrgpr genes (Mrgpr-clusterΔ−/− or Mrgpr-cluster KO) with bilirubin (Liu et al., 2009). Mrgpr-cluster KO animals scratched approximately 75% less than wild type (WT) mice, indicating that one or more of the 12 Mrgprs within the cluster mediates bilirubin-induced pruritus (Figure 1C). To identify which Mrgpr is sensitive to bilirubin, we individually expressed each of the 12 Mrgprs deleted in the Mrgpr-cluster KO mouse in human embryonic kidney (HEK) 293 cells and monitored changes in intracellular calcium upon applying bilirubin. To ensure we would observe a calcium response following a true ligand-receptor interaction, we expressed the receptors in HEK293 cells stably expressing the G-protein alpha-subunit Gα15, a Gα protein that couples GPCRs to intracellular calcium stores via phospholipase C (PLC). Among the twelve cell lines expressing an Mrgpr, only MRGPRA1-expressing cells exhibited a calcium response to bilirubin (EC50 of 145.9 µM (Alemi et al., 2013)) (Figure 2A,D). The same cells that responded to bilirubin also responded to FMRF, an MRGPRA1 agonist (Dong et al., 2001). To ensure that bilirubin initiated signaling at MRGPRA1 and not downstream, we pre-treated MRGPRA1-expressing cells with inhibitors of GPCR signaling: the PLC inhibitor U73122 or the Gαq inhibitor YM-254890. Both compounds abolished bilirubin-induced calcium responses (Figure 2B–C). Figure 2 with 2 supplements see all Download asset Open asset Bilirubin activates murine MRGPRA1 and human MRGPRX4. Ca2+ imaging and transformed binding isotherms of HEK293 cells stably expressing MRGPRA1 (A-E) or MRGPRX4 (F-J). (A-C, F-H) 50 µM bilirubin was added where indicated by black bars. After 15 s, a 1 min wash was applied. Mean ±95% confidence interval (CI) depicted. n = 10. In (A) 30 µM FMRF was added after washing as indicated by the black bar. In (B-C) and (G-H), cells were pre-incubated with either 10 µM of the PLC inhibitor U73122 or 10 µM of the Gαq inhibitor YM254890 for 30 min prior to imaging. Concentration–Ca2+ response curves of bilirubin, conjugated bilirubin, and hemin towards (D) MRGPRA1, (I) MRGPRX4, and (N) MRGPRC11 and BAM8-22 towards MRGPRC11, an established peptide ligand. Data are a representative experiment of three independent replicates performed in triplicate, depicted as mean ±s.e.m. Transformed binding isotherms for bilirubin, conjugated bilirubin, and hemin to (E) MRGPRA1, (J) MRGPRX4, and (O) MRGPRC11 and BAM8-22 to MRGPRC11. Data are an average of three independent experiments, depicted as mean ±s.e.m. (K) Bilirubin-stimulated G-protein activity of partially-purified MRGPRA1, MRGPRX4, and MRGPRC11 membrane complexes. [35S]GTPγS binding was measured in the presence of 0.5% DMSO or 50 μM bilirubin. Mean ±s.e.m. depicted. **, p < 0.01; two-tailed unpaired Student’s t-test. (L) Scratching bouts from injection of 60 µg of bilirubin in WT and A1 KO animals. WT n = 10, A1 KO n = 12. (M) Scratching bouts from injection of 60 µg of FMRF in WT and A1 KO animals. WT n = 8, A1 KO n = 7. (L-M) Mean ±s.e.m. depicted. Open circles represent individual mice. *, p< 0.05 by two-tailed unpaired Student’s t-test. https://doi.org/10.7554/eLife.44116.006 Figure 2—source data 1 Source data for Figure 2 . https://doi.org/10.7554/eLife.44116.009 Download elife-44116-fig2-data1-v3.xlsx In addition to bilirubin, glucuronidated bilirubin is often upregulated in jaundice-associated itch. We assessed whether ditaurate bilirubin, a distinct but similar bilirubin derivative, could activate MRGPRA1. Indeed, ditaurate bilirubin activated MRGPRA1-expressing cells (Figure 2D). Hemin failed to activate MRGPRA1 (Figure 2D), consistent with our earlier behavioral findings in which hemin did not evoke scratching. No other Mrgpr among the 12 that we screened responded to bilirubin (Figure 2N, Figure 2—figure supplement 1). The human MRGPRX family of receptors has functional similarities between species but have no obvious structural homologs in rodents (Solinski et al., 2014; Zylka et al., 2003). The mouse Mrgpra family is closest in sequence homology to the human MRGPRX family (Dong et al., 2001; Lembo et al., 2002; Zhang et al., 2005). Of the four human MRGPRX receptors, only MRGPRX4-expressing cells responded to bilirubin (EC50 of 61.9 µM (Azimi et al., 2017)) (Figure 2F,I). U73122 and YM-254890 inhibited bilirubin-induced calcium responses in MRGPRX4-expressing cells just as with MRGPRA1 (Figure 2G–H). Conjugated bilirubin also activated MRGPRX4, whereas hemin had no effect (Figure 2I). To confirm that bilirubin directly binds the identified receptors, we assayed thermophoresis of each receptor in the presence and absence of bilirubin. Thermophoresis of a molecule is affected by physical parameters such as size, charge, and solvation. By extension, the thermophoresis of one molecule is altered when it interacts with another, and can therefore be used to measure interactions between molecules (Duhr and Braun, 2006). Using this approach, we determined that bilirubin bound MRGPRA1 with a KD of 92.9 ± 15 µM and MRGPRX4 with a KD of 54.4 ± 13 µM (Figure 2E,J). Bilirubin exhibited little to no affinity for the closely related BAM8-22 receptor MRGPRC11 (Figure 2O). Hemin, which did not activate MRGPRA1 or MRGPRX4 by calcium imaging (Figure 2D,I), also did not bind MRGPRA1 or MRGPRX4 (Figure 2E,J). Conjugated bilirubin bound both MRGPRA1 and MRGPRX4, although with a lower affinity than unconjugated bilirubin (Figure 2E,J). To make certain that bilirubin activates MRGPRA1 and MRGPRX4 upon binding, we measured exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP), one of the first events in GPCR signaling. Bilirubin increased GTP binding to MRGPRA1 and MRGPRX4 membrane complexes, but not to MRGPRC11 (Figure 2K). To confirm that bilirubin activates MRGPRA1 in vivo to trigger itch, we generated an Mrgpra1 (A1 KO) knockout mouse line using CRISPR-Cas9 (Jinek et al., 2012) (Figure 2—figure supplement 2). A1 KO animals scratched significantly less than WT mice after exposure to either bilirubin or the established agonist FMRF, demonstrating that Mrgpra1 is functional in adult mice (Figure 2L–M). The KD of bilirubin towards MRGPRA1 and MRGPRX4 suggests that bilirubin likely does not interact with these receptors in healthy individuals. Additional ligands with nanomolar affinities towards MRGPRA1 or MRGPRX4 may exist that modulate the receptors in normal physiology. We reasoned that if bilirubin triggers itch through MRGPRA1 and MRGPRX4, bilirubin should activate these receptors in sensory itch neurons. Previous studies have demonstrated that both Mrgpra1 and MRGPRX4 are expressed in sensory neurons within the dorsal root ganglia (DRG) (Dong et al., 2001; Flegel et al., 2015; Lembo et al., 2002). Mrgpra1 is expressed in a subset of adult DRG and trigeminal ganglia (TG) sensory neurons that innervate the skin and ramify in lamina I and II of the spinal cord (Figure 3A–D). Bilirubin elicited robust action potentials in small-diameter (<30 µm) WT DRG sensory neurons at a proportion consistent with the percentage of sensory neurons that encode itch (5 of 50). Bilirubin failed to elicit action potentials in A1 KO neurons (0 of 60), suggesting bilirubin activates sensory neurons through MRGPRA1 (Figure 3E). Bilirubin-sensitive neurons had an average somal diameter of 20.4 ± 1.3 µm, a diameter characteristic of itch sensory neurons (Figure 3F). Applying bilirubin to neurons elicited calcium transients in approximately 5% of WT DRG neurons (Figure 3G), whereas significantly fewer sensory neurons from either Mrgpr-cluster KO or A1 KO mice responded (Figure 3H). We sought to determine whether expression of either MRGPRA1 or MRGPRX4 was sufficient to render neurons sensitive to bilirubin. To address this question, we infected Mrgpr-cluster KO DRGs with lentivirus carrying either Mrgpra1, MRGPRX4, or MRGPRX3. Bilirubin activated 14% of Mrgpra1-and 32% of MRGPRX4-transduced Mrgpr-cluster KO DRGs (Figure 3I–J). Mrgpr-cluster KO DRGs infected with the control gene MRGPRX3 did not respond to bilirubin. Figure 3 Download asset Open asset Bilirubin activates sensory neurons in an MRGPR-dependent manner. (A–D) Confocal microscopy immunofluorescence images of adult mouse tissue sections from Mrgpra1GFP animals with GFP expression under the control of the endogenous Mrgpra1 locus. (A) Mrgpra1 expression in dorsal root ganglia. Green depicts Mrgpra1GFP. Red depicts anti-PLAP antibody staining where PLAP expression is controlled by the endogenous Mrgprd locus (MrgprdPLAP). Blue depicts antibody staining against calcitonin gene-related peptide (CGRP). Scale bar is 50 µM. (B) Trigeminal ganglia (TG) stained with Mrgpra1GFP (green) and anti-Substance P antibody (red). Scale bar is 50 μm. (C) Back skin stained with anti-GFP antibody (green) to visualize Mrgpra1GFP nerve fibers in the dermis. Blue represents DAPI counterstain. Scale bar is 50 μm. (D) Spinal cord (SC) (lamina 1 and 2) stained with anti-GFP and IB4-564. Mrgpra1GFP (green) staining was found in lamina two along with IB4 (red) positive terminals. Scale bar is 100 μm. (E) Representative whole-cell current-clamp recording of either WT or A1 KO DRG neurons. In WT DRG, bilirubin elicited action potentials in 5 out of 50 small-diameter neurons. In A1 KO DRG, bilirubin elicited action potentials in 0 out of 60 small-diameter neurons. Fisher’s exact test p < 0.05. (F) Histogram of bilirubin-activated neuronal soma diameter. (G) Ca2+ imaging of WT DRG neurons. Mean ±95% depicted. where indicated by black bars. After a 10 50 µM bilirubin was 50 was added at the end of each n = neurons. of A1 and Mrgpr-cluster KO DRG by and 50 µM bilirubin. *, P < 0.05; **, p < 0.01; ***, p < 0.001; was to be activated if for at a 30 (I) Ca2+ imaging of Mrgpr-cluster KO DRG neurons after either with lentivirus = or infected with lentivirus Mrgpra1 = 6), MRGPRX4 = or MRGPRX3 = 50 µM bilirubin was added when indicated by the black bar. After s, a 1 min wash was before addition of 50 where indicated by black bars. Mean ±95% depicted. n = 10 neurons. (J) of and neurons by bilirubin. ***, p < Figure data 1 Source data for Figure Download neurons with neurons that responded to 1 a for that itch sensory neurons (Han et al., (Figure To that bilirubin activates itch we performed calcium imaging on DRG neurons isolated from mice, which the protein in neurons. Bilirubin activated a percentage of neurons (Figure To confirm that bilirubin activates sensory neurons in we injected 5 of or bilirubin into of mice, which the calcium in DRG sensory neurons et al., 2014). but not activated DRG sensory neurons in the of mice (Figure receptor and other Ca2+ with bilirubin from sensory neurons (Figure et al., 2009; Liu et al., 2009; et al., Figure Download asset Open asset Bilirubin activates a of sensory neurons in a (A) representative whole-cell current-clamp recording of a WT DRG to addition of both 50 µM bilirubin and 1 chloroquine (B) Ca2+ imaging of WT DRG neurons. Mean ±95% depicted. where indicated by black bars. After a 10 50 µM bilirubin was After s, a 3 min wash was before 1 chloroquine was After 15 s, 50 was n = 10 neurons. (C) of neurons activated by either bilirubin chloroquine = 7, Chloroquine = = (D) of neurons as assessed by calcium imaging with 1 or 50 µM bilirubin. (E) In vivo Ca2+ imaging of animals. a was performed to were before a injection in and injection of 5 of 100 μM bilirubin. is a representative Ca2+ imaging of bilirubin-activated n = identified by imaging The black is the mean and lines represent confidence intervals. (F) were with 10 µM for 10 min before of 50 µM bilirubin. (G) of WT with either or 10 µM by 50 µM bilirubin. Figure data 1 Source data for Figure Download We whether chronic of bilirubin in like in cholestasis, Mrgpr-dependent itch. is the primary by which bilirubin is and patients with elevated levels of bilirubin and other pruritogenic substances in their blood (Alemi et al., To induce and cholestasis, we to mice et al., We Mrgpr-cluster and A1 KO animals with for before itch (Figure Mrgpr-cluster and A1 KO animals exhibited by in plasma bilirubin, bile and (Figure Figure supplement Figure 5 with 3 supplements see all Download asset Open asset Mrgpr-cluster and KO animals decreased pruritus. (A) for of (B) Scratching bouts for and mice among Mrgpr-cluster and A1 KO were assessed in a 30 min the WT n = 15, Mrgpr-cluster KO n = A1 KO n = WT n = Mrgpr-cluster KO n = A1 KO n = (C) Scratching bouts for and animals among WT and KO were assessed in a 30 min the WT n = 5 and KO n = WT n = and KO n = (D) bilirubin levels from WT and Mrgpr-cluster KO and animals. the WT n = Mrgpr-cluster KO n = the WT n = Mrgpr-cluster KO n = (E) bile acid levels from and animals. the WT n = Mrgpr-cluster KO n = 5, KO n = the WT n = 10, Mrgpr-cluster KO n = 7, KO n = (F) for of (G) Scratching bouts for and WT and A1 KO animals. the n = 5 for WT n = 10 and A1 KO n = Scratching bouts from and WT and KO animals. the n = WT n = and KO n = 7. (I) of serum bilirubin levels from animals and scratching of = (J) of skin bilirubin levels from animals and scratching of = (K) of skin and serum bilirubin levels from animals. of = Mean ±s.e.m. depicted. Open circles represent individual data *, p < 0.05; **, p < 0.01; ***, p < by unpaired two-tailed Student’s t-test. Figure data 1 Source data for Figure Download significantly increased pruritus in all animals (Figure However, Mrgpr-cluster KO and A1 KO mice scratched less than WT mice (Figure suggesting that MRGPRA1 mediates a of hepatobiliary pruritus. In bile endogenous and are often increased in and have been to mediate pruritus (Alemi et al., 2013; et al., et al., et al., The serum of animals exhibited elevated bilirubin and bile (Figure whereas neither the endogenous peptide and and nor the enzyme were elevated (Figure supplement 1E–F). To whether other at MRGPRs in mice, we injected WT and Mrgpr-cluster KO with acid bile and other elicited of scratching in WT and Mrgpr-cluster KO animals (Figure supplement Mrgprs are receptors. should be noted that there bile and which that may be of Based on this data, we that Mrgpr-cluster KO and A1 KO mice scratched less with because MRGPRA1 mediates bilirubin-induced itch. To determine whether bilirubin is MRGPRA1 to stimulate itch in cholestasis, we in a mouse that the enzyme for bilirubin, biliverdin KO) and (Figure Figure supplement KO mice lack Blvra, the gene that encodes for bilirubin KO mice do not have levels of bilirubin in plasma (Figure supplement with KO mice scratched significantly less than WT mice (Figure levels of bile and were between KO animals and WT (Figure supplement response to is not due to itch as KO mice scratched when injected with either chloroquine or bilirubin (Figure supplement To confirm that the differences in pruritus were not just to we the to A1 and KO mice et al., We mice with either 50 or for before itch (Figure itch in WT whereas A1 KO and KO mice scratched significantly less than WT mice (Figure Notably, we found that plasma bilirubin with itch in patients and in animals (Figure We that the levels of bilirubin in the skin would better with itch than serum bilirubin because bilirubin likely binds and activates the sensory neurons in the skin. Unlike with skin bilirubin to be a much