Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice  Sang Hoon Lee, PhD, Pyung Sun Cho, PhD, Raquel Tonello, PhD, Han Kyu Lee, BS, Jun Ho Jang, PhD, Gi Yeon Park, BS, Sun Wook Hwang, PhD, Chul-Kyu Park, PhD, Sung Jun Jung, MD, PhD, Temugin Berta, PhD  Journal of Allergy and Clinical Immunology  Volume 142, Issue 4, Pages 1349-1352.e16 (October 2018) DOI: 10.1016/j.jaci.2018.05.031 Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Serotonergic antidepressants trigger scratching behavior in mice through HTR2B activation in sensory neurons. A and B, Subcutaneous injections of 100 μmol/L to 1 mmol/L sertraline into the cheek do not evoke wiping behavior (Fig 1, A) but do elicit dose-dependent scratching behaviors (Fig 1, B; n = 5 per group). C, Time course of scratching behavior to sertraline (1 mmol/L) shows a rapid scratching response (n = 5 per group). D, Skin 5-HT levels are not increased 10 minutes after injection with sertraline (1 mmol/L, n = 4 per group). E, Scratching behaviors evoked by sertraline, citalopram, and fluoxetine (all at 1 mmol/L) are increased compared with those in vehicle-treated mice and decreased in mice that are coinjected with RS127445 (40 μg, n = 4-5 mice/group). F, Scratching behavior evoked by α-Me-5HT (10 μg) is decreased in mice coinjected with RS127445 (40 μg, n = 5 mice/group). G and H, Sertraline-induced scratching is attenuated in mice after knockdown of HTR2B in DRG neurons or in mice treated with resiniferatoxin (RTX) compared with their respective vehicle-treated control animals (n = 5-6). Data are means ± SEMs. *P < .05, **P < .01, and ***P < .001. NS, Not significant. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 HTR2B activation induces neuronal and scratching behaviors through TRPC4 in sensory neurons. A, Representative Fura-2 ratio in response to α-Me-5HT (5 μmol/L) and capsaicin (Caps; 1 μmol/L) in cultured DRG neurons. B, Representative Fura-2 images of DRG-cultured neurons from wild-type (WT), TRPV1 1 KO, and TRPC4 KO mice in response to vehicle control, α-Me-5HT (5 μmol/L), and capsaicin (1 μmol/L). Scale bar = 10 μm. C, Scratching behavior evoked by α-Me-5HT (10 μg) is similar in WT and TRPV1 KO mice but diminished in TRPC4 KO mice (n = 4-5 mice/group). D and E, This behavior is also attenuated by the TRPC4 antagonist ML204 (40 μg, n = 5 per group; Fig 2, D) and knockdown of TRPC4, but not TRPC1 and TRPC5, in DRG neurons (n = 5-17 mice/group; Fig 2, E). F, Sertraline, citalopram, and fluoxetine (all at 1 mmol/L)–evoked scratching behaviors are decreased in mice coinjected with ML204 (40 μg, n = 5-6 mice/group). G, Sertraline-evoked scratching behavior is abolished in TRPC4 KO mice (n = 6 mice/group). H, Model of action for SSRIs and α-Me-5HT through HTR2B and TRPC4 in sensory neurons, leading to pruritus. Data are means ± SEMs. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Sertraline and serotonin trigger itch through HTR2B. A, Scratching behavior evoked by the SSRI sertraline (1 mmol/L) is not attenuated by the HTR7 antagonist SB269970 (40 μg, n = 6 mice/group). B, Similar to the HTR2B antagonist RS127445, scratching behavior evoked by sertraline (1 mmol/L) is decreased significantly in mice coinjected with SB204741, another HTR2B antagonist (40 μg, n = 6 mice/group). C, Scratching behavior evoked by serotonin (10 μg) is decreased significantly in mice coinjected with RS127445 (n = 5 mice/group). Error bars indicate means ± SEMs. *P < .05, **P < .01, and ***P < .001. NS, Not significant. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 α-Me-5-HT triggers itch and action potential firing through HTR2B. A, Subcutaneous injections of α-Me-5HT into the cheek do not evoke wiping behavior but elicit strong scratching behaviors. B, Time course of scratching behaviors (n = 5 mice/group). C, Example of ex vivo single-fiber recordings in application of α-Me-5HT (200 mmol/L) or α-Me-5HT with RS127445 (800 mmol/L, n = 9 fibers over 3 mice). Error bars indicate means ± SEMs. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 siRNA reduces HTRB2 in DRGs, and resiniferatoxin (RTX) treatment impairs capsaicin-evoked pain. A and B, Western blotting showing knockdown of HTR2B expression of approximately 40% in DRGs by Htr2b siRNA treatment (3 μg/d for 3 days). C, After pretreatment with RTX (50 μg/kg, single injection), mice are completely insensitive to capsaicin-evoked pain (n = 5 mice/group). Error bars indicate means ± SEMs. *P < .05 and ***P < .001. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 HTRB2 is expressed in small TRPV1+ sensory neurons. A, Immunohistochemistry in mouse TGs and DRGs shows that HTR2B is enriched in small- to medium-sized cells (cross-sectional area in square micrometers, n = 4-5 sections, 791 DRG neurons). Error bars indicate means ± SEMs. Scale bars = 20 μm. B, HTR2B is colocalized with the peptidergic fiber marker CGRP but not with the non-peptidergic fiber marker IB4 in DRG neurons. C, Single-cell RT-PCR showing that all small-sized DRG neurons (diameter, <25 μm) expressing Htr2b also express Trpv1. M, Molecular weight; NC, negative controls from pipettes that did not harvest any cell contents but were submerged in the bath solution. Yellow asterisk indicates HTR2B- positive neurons. Full-length gels are shown in Fig E5. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Full-length gels of Fig E4, C. Single-cell RT-PCR analysis in small DRG neurons shows colocalization of HTR2B with TRPV1. Red boxes show representative results for Fig E4, C. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 α-Me-5HT evoked calcium responses through phospholipase C (PLC). A and B, Ca2+ transients evoked by sequential application of 5 μmol/L α-Me-5HT were blocked by extracellular Ca2+-free conditions (n = 7; Fig E6, A) but not by 1 μmol/L thapsigargin (n = 9; Fig E6, B). C, α-Me-5HT (5 μmol/L)–induced Ca2+ responses were abolished by 2 μmol/L U73122 (n = 14) but not by U73343 (n = 6). Error bars indicate means ± SEMs. ****P < .0001. NS, Not significant. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 α-Me-5HT and sertraline evoked itch responses independently from TRPA1 and TRPV4. A-C, Scratching behavior evoked by α-Me-5HT (4 μg) is significantly greater compared with that evoked by a vehicle control (3.8 ± 2.154 bouts per 30 minutes) but is not attenuated in TRPA1 KO mice (TRPA−/−) compared with wild-type (WT) mice (n = 5 per group; Fig E7, A) or by the TRPA1 antagonist HC030031 (40 μg, n = 6 mice/group; Fig E7, B) and the TRPV4 antagonist HC067047 (40 μg, n = 6 mice/group; Fig E7, C). D, Similarly, scratching behavior evoked by sertraline (1 mmol/L) is unaffected in TRPA−/− mice compared with WT mice (n = 6 mice/group). Of note, vehicle control induced very limited scratching behavior. Error bars indicate means ± SEMs. *P ≥ .05. NS, Not significant. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E8 TRPC4 colocalizes with HTR2B and regulates neuronal activity induced by HTR2B agonists. A, Immunohistochemistry showing that HTR2B colocalizes with TRPC4 in TG. DAPI, 4′-6-Diamidino-2-phenylindole dihydrochloride. B, Calcium responses evoked by HTR2B agonist BW-723C86 (5 μmol/L) in a wild-type (WT) mouse (25/122 neurons). In contrast, no calcium response was observed in a TRPC4 KO mouse (TRPC4−/−, 0/103 neurons). Traces are representative of Fura-2 calcium responses, and the histogram shows the percentage of positive cells to BW-723C86. C, α-Me-5HT (patch, 2 μmol/L)–induced currents were abolished by ML204 (patch, 20 μmol/L). D, Example of ex vivo single-fiber recordings in application of α-Me-5HT (666 μmol/L) or α-Me-5HT with the TRPC4 antagonist ML204 (3.5 mmol/L, n = 14 fibers over 3 mice). Error bars indicate means ± SEMs. **P < .01 and ****P < .0001. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E9 HTR2B activation promotes scratching behavior through TRPC4. A, Time course of scratching behavior evoked by α-Me-5HT (10 μg) is dose dependently decreased by the TRPC4 antagonist ML204 (n = 5 mice/group). B, Quantification of total bouts per 30 minutes of scratching behavior evoked by α-Me-5HT (10 μg) and its dose-dependent attenuation by ML204 (n = 5 mice/group). C, Scratching behavior evoked by the HTR2B agonist BW-723C86 (4 μg) is significantly decreased in mice coinjected with ML204 (40 μg, n = 6 per group). D, Scratching behavior evoked by BW-723C86 (10 μg) was also abolished in TRPC4 KO mice compared with wild-type (WT) mice (n = 5 mice/group). Error bars indicate means ± SEMs. **P < .01 and ****P < .0001. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E10 HTR2B activation induces itch through TRPC4 but not TRPC1 and TRPC5. A, Onset of scratching behavior evoked by α-Me-5HT (10 μg) is significantly decreased in mice injected with TRPC4 siRNA (siTRPC4, n = 6-7 mice/group). B-D, Western blotting showing knockdown of TRPC4, TRPC1, and TRPC5 expression of approximately 40% to 60% in DRGs by targeting siRNA treatment (3 μg/d, 3 days, n = 4-5 mice/group). Error bars indicate means ± SEMs. *P < .05, **P < .01, and ***P < .001. Full-length gels are shown in Fig E11. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E11 Full-length gels of Figs E3, A, and E10, B-D. Western blot analysis of DRG tissues shows knockdown of HTR2B, TRPC4, TRPC1, and TRPC5. Red boxes show representative results for Figs E3, A, and E10, B-D. N, Control siRNA; S, targeting siRNA. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E12 HTR2B and TRPC4 are expressed in human DRG tissues. PCR amplification of Htr2b (A) and Trpc4 (B) transcripts shows expression in mouse and human DRGs. Experimental details and primer sequences are described in the Methods section. Journal of Allergy and Clinical Immunology 2018 142, 1349-1352.e16DOI: (10.1016/j.jaci.2018.05.031) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions