The CysLT2R receptor mediates leukotriene C4-driven acute and chronic itch

Abstract

Acute and chronic itch are burdensome manifestations of skin pathologies including allergic skin diseases and atopic dermatitis, but the underlying molecular mechanisms are not well understood. Cysteinyl leukotrienes (CysLTs), comprising LTC₄, LTD₄, and LTE₄, are produced by immune cells during type 2 inflammation. Here, we uncover a role for LTC₄ and its signaling through the CysLT receptor 2 (CysLT₂R) in itch. Cysltr2 transcript is highly expressed in dorsal root ganglia (DRG) neurons linked to itch in mice. We also detected CYSLTR2 in a broad population of human DRG neurons. Injection of leukotriene C₄ (LTC₄) or its nonhydrolyzable form NMLTC₄, but neither LTD₄ nor LTE₄, induced dose-dependent itch but not pain behaviors in mice. LTC₄-mediated itch differed in bout duration and kinetics from pruritogens histamine, compound 48/80, and chloroquine. NMLTC₄-induced itch was abrogated in mice deficient for Cysltr2 or when deficiency was restricted to radioresistant cells. Itch was unaffected in mice deficient for Cysltr1, Trpv1, or mast cells (W^Sh mice). CysLT₂R played a role in itch in the MC903 mouse model of chronic itch and dermatitis, but not in models of dry skin or compound 48/80- or Alternaria-induced itch. In MC903-treated mice, CysLT levels increased in skin over time, and Cysltr2^-/- mice showed decreased itch in the chronic phase of inflammation. Collectively, our study reveals that LTC₄ acts through CysLT₂R as its physiological receptor to induce itch, and CysLT₂R contributes to itch in a model of dermatitis. Therefore, targeting CysLT signaling may be a promising approach to treat inflammatory itch.

Keywords: atopic dermatitis; inflammation; itch; neuroimmune; skin.

Fig. 1.

Cysltr2 is expressed on a subset of DRG sensory neurons. (A) Diagram of the LT pathway. (B and C) Expression of selected transcripts (B, related to itch; C, LT receptors) in mouse DRG neuron populations clustered into functional subsets based on single-cell RNA-seq data. Full dataset and methods are available in a previous study (32). (D) Triple/double-label ISH done with RNAscope in DRG (Left, Cysltr2, red; Nppb, blue; Hrh1, green; Middle, Cysltr2, red; MrgprA3, green; Mrgprd, blue; Right, Cysltr2, red; Trpv1, green). (Scale bar: 50 µm.) (E and F) ISH quantification: (E) Venn diagram representing overlap between markers and (F) percentages of Cysltr2⁺ neurons within subsets defined by other markers (n = 3 mice). (G) Representative images of human DRG labeled with RNAscope ISH for CYSLTR2 (red), NPPB (green), and TRPV1 (blue) and costained with DAPI (cyan). Lipofuscin (globular structures) that autofluoresced in all three channels and appear white in the overlay image were not analyzed, as this is background signal that is present in all human nervous tissue. (Scale bar: 50 μm.) (H) Pie chart displaying the distribution of CYSLTR2 neuronal subpopulations in human DRG. (I) Percentage of all human sensory neurons expressing CYSLTR2 transcript (Top) and of TRPV1 neurons and NPPB neurons coexpressing CYSLTR2 (Bottom). Values presented as mean ± SEM.

Fig. 2.

LTC₄ but not LTD₄ or LTE₄ induces dose-dependent acute itch behaviors. (A) Diagram of experimental design of acute itch/pain induction by intradermal injection of ligands in the cheek of the mice. (B) Scratching bouts in response to vehicle, LTC₄, NMLTC₄, and LTD₄ at 0.6 nmol (n = 8 to 11). (C) Wiping responses, indicative of pain, to vehicle, LTC₄, NMLTC₄, and LTD₄ at 0.6 nmol (n = 8 to 11). (D) Scratching bouts in response to vehicle, NMLTC₄, and LTE₄ at 0.6 nmol (n = 4 to 8). (E) Wiping responses, indicative of pain, to vehicle, LTE₄, and NMLTC₄ at 0.6 nmol (n = 4 to 8). (F) Scratching bouts’ dose responses to NMTLC₄ and LTD₄ were recorded for 30 min at different concentrations and scored live (n = 3 to 12). (G) Scratching bouts’ dose responses to LTC₄ and NMLTC₄ were recorded for 30 min at different concentrations (n = 6 to 8). (H and I) Analysis of kinetics and scratching bout duration differences in response to various pruritogens: LTC₄ (0.6 nmol), IL-31 (0.02 nmol), histamine (100 μg), compound 48/80 (100 μg), CQ (200 μg), and vehicles (phosphate-buffered saline [PBS] and dimethyl sulfoxide [DMSO]). (H) Absolute numbers of bouts shorter than 0.3 s, bouts between 0.3 and 1 s, and bouts longer than 1 s. (I) Distribution of bouts according to their duration. Values presented as mean ± SEM. One-way ANOVA with Dunnett’s posttest. ns, nonsignificant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 3.

LTC₄-induced itch is dependent on CysLT₂R. (A and B) Scratching bouts induced by intradermal cheek injection of vehicle or LTC₄ 0.6 nmol in B6 mice or by injection of LTC₄ 0.6 nmol in Cysltr2^−/− mice (7 to 44 wk old; A) and detailed scratching bout kinetics over 30 min (^#B6 + LTC₄ vs. B6 + vehicle; *B6 + LTC₄ vs. Cysltr2^−/− + LTC₄; B). (C and D) Scratching bouts induced by intradermal cheek injection in Cysltr2^+/+ or Cysltr2^−/− mice of NMLTC₄ (0.6 nmol; C) and detailed scratching bout kinetics over 45 min (D). (E and F) Generation of BM chimeras with WT or Cysltr2^−/− mice as donors and WT or Cysltr2^−/− mice as recipients. (E) Diagram of BM transplant procedure. (F) Scratching bouts induced by intradermal cheek injection of NMLTC₄ 0.6 nmol in naïve mice and in BM chimera. Behavior recorded in daylight settings (not in iBOB). (G–I) Scratching bouts induced by intradermal cheek injection of NMLTC₄ (0.6 nmol) in (G) Kit^Wsh/Wsh mice, (H) Cysltr1^−/− mice, and (I) Trpv1^−/− mice. (J) Scratching bouts induced by the coinjection of NMLTC₄ (0.6 nmol) and LTD₄ (2 nmol) in B6 mice and in Cysltr1^−/− mice. Values presented as mean ± SEM. Unpaired t test (C, G, and I), repeated-measures two-way ANOVA with Šidák’s posttest (B and D), one-way ANOVA with Dunnett’s posttest (F), and one-way ANOVA with Tukey’s posttest (A and J). ns, nonsignificant (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig. 4.

CysLT levels are increased in the skin of the MC903 model of dermatitis and itch. (A) Diagram of procedure: daily application of MC903 on the mouse ear for 12 d. (B) Scratching bouts recorded on days 3, 10, and 12 for 60 min before daily application of vehicle (ethanol) or MC903. (C) Percentage of ear thickness change following daily application of vehicle or MC903. (D) H&E staining performed on ear section at day 12 of MC903 model. (Scale bar: 50 µm.) (E) CysLTs (LTC₄, LTD₄, and LTE₄) levels measured by ELISA at days 3, 6, 10, and 12 (n = 5 to 6). (F and G) Liquid chromatography–mass spectrometry. (F) Representative chromatograms for LTC₄ (Left) and LTE₄ (Right) from ear homogenates at day 12 after daily vehicle treatment (Top) or MC903 treatment (Bottom). (G) LTC₄ and LTE₄ quantification per milligram of ear collected at day 12 after vehicle or MC903 treatment. Values presented as mean ± SEM. Repeated-measures two-way ANOVA, Šidák’s posttest (B and C), two-way ANOVA with Šidák’s posttest (E), and unpaired t test (G). ns, nonsignificant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig. 5.

CysLT₂R is involved in chronic itch but not in inflammation in MC903. (A) Scratching bouts recorded on days 3, 6, 10, and 12 for 60 min before daily application of MC903 in Cysltr2^+/+ or Cysltr2^−/− mice. (B) Bout duration analysis for Cysltr2^+/+ and Cysltr2^−/− mice at day 12 of MC903 (number of bouts: <0.3 s, between 0.3 and 1 s, and >1 s). (C) Percentage of ear thickness change following daily application of MC903 in Cysltr2^+/+ or Cysltr2^−/− mice. (D and E) H&E staining performed on ear section at day 12 of the MC903 model in Cysltr2^+/+ or Cysltr2^−/− mice. (D) Representative images of H&E staining. (E) Quantification of epidermal thickness (n = 8 to 9 with 10 to 15 fields quantified per animal). (F) Quantification of immune cell populations by flow cytometry from ear homogenates collected at day 12 from Cysltr2^+/+ or Cysltr2^−/− mice (n = 3). Values presented as mean ± SEM. Repeated-measures two-way ANOVA, Šidák’s posttest (A and C), and unpaired t test (B, E, and F). ns, nonsignificant (*P < 0.05; **P < 0.01; ****P < 0.0001).