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. 2016 Nov 22;113(47):E7572-E7579.
doi: 10.1073/pnas.1606608113. Epub 2016 Nov 7.

IL-33/ST2 signaling excites sensory neurons and mediates itch response in a mouse model of poison ivy contact allergy

Boyi Liu  1   2 , Yan Tai  2 , Satyanarayana Achanta  2 , Melanie M Kaelberer  2 , Ana I Caceres  2 , Xiaomei Shao  3 , Jianqiao Fang  3 , Sven-Eric Jordt  4
Affiliations

IL-33/ST2 signaling excites sensory neurons and mediates itch response in a mouse model of poison ivy contact allergy

Boyi Liu et al. Proc Natl Acad Sci U S A. .

Abstract

Poison ivy-induced allergic contact dermatitis (ACD) is the most common environmental allergic condition in the United States. Case numbers of poison ivy ACD are increasing due to growing biomass and geographical expansion of poison ivy and increasing content of the allergen, urushiol, likely attributable to rising atmospheric CO2 Severe and treatment-resistant itch is the major complaint of affected patients. However, because of limited clinical data and poorly characterized models, the pruritic mechanisms in poison ivy ACD remain unknown. Here, we aim to identify the mechanisms of itch in a mouse model of poison ivy ACD by transcriptomics, neuronal imaging, and behavioral analysis. Using transcriptome microarray analysis, we identified IL-33 as a key cytokine up-regulated in the inflamed skin of urushiol-challenged mice. We further found that the IL-33 receptor, ST2, is expressed in small to medium-sized dorsal root ganglion (DRG) neurons, including neurons that innervate the skin. IL-33 induces Ca2+ influx into a subset of DRG neurons through neuronal ST2. Neutralizing antibodies against IL-33 or ST2 reduced scratching behavior and skin inflammation in urushiol-challenged mice. Injection of IL-33 into urushiol-challenged skin rapidly exacerbated itch-related scratching via ST2, in a histamine-independent manner. Targeted silencing of neuronal ST2 expression by intrathecal ST2 siRNA delivery significantly attenuated pruritic responses caused by urushiol-induced ACD. These results indicate that IL-33/ST2 signaling is functionally present in primary sensory neurons and contributes to pruritus in poison ivy ACD. Blocking IL-33/ST2 signaling may represent a therapeutic approach to ameliorate itch and skin inflammation related to poison ivy ACD.

Keywords: IL-33; allergic contact dermatitis; cytokine; itch; pain.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Mouse transcriptome microarray analysis of oxazolone- or urushiol-challenged mouse skin. (A) Scheme of treatment in urushiol- or oxazolone-induced mouse ACD model. (B) Heat map showing top 15 most up-regulated inflammatory cytokines and chemokines in oxazolone (Oxa)- and urushiol (Uru)-challenged mouse neck skin, identified by mouse transcriptome microarray analysis. Vehicle group (Veh) mice were treated with acetone. n = 3 mice per group. (C) Fold changes of IL-33 gene transcript in skin samples from oxazolone- and urushiol-challenged mouse by qPCR. (D and E) IL-33 from skin and plasma of mice by ELISA. n = 7 or 8 mice per group. ND, not detectable. (F) Immunofluorescence images of IL-33 staining (green) in mouse neck skin from frozen sections. Nuclei were labeled with DAPI (blue). (G) Summary of IL-33 immunostaining in F. n = 7 or 8 mice per group. (H) Double immunostaining showing the overlapping of IL-33 with keratin 14 in the skin of urushiol-induced ACD mice. *P < 0.05; **P < 0.01; ***P < 0.001 vs. vehicle/control group. One-way ANOVA followed by Tukey post hoc test was used for statistical analysis. (Scale bars, 20 µm.)
Fig. 2.
Fig. 2.
Analysis of IL-33 receptor ST2 expression in DRG neurons. (A and B) Summary of gene expression levels of IL-33 receptor complex IL-1RAcP and ST2 in human (A) and mouse (B) DRGs. Mouse β-actin and human GAPDH were used as housekeeping genes. (C) Immunostaining showing the immunoreactivity of IL-33 receptor ST2 (green) in mouse cervical DRG neurons (identified by Nissl staining; red). (Magnification: 60×.) (D) Cell size distribution frequency of ST2-positive and Nissl staining-positive neurons (636 neurons/12 inconsecutive sections/5 mice). (E) ST2 is expressed in cutaneous Fast Blue (purple) labeled DRG neurons indicated by white arrows. (Scale bars, 20 µm.)
Fig. S1.
Fig. S1.
Antigenic peptide preabsorption blocks ST2 antibody staining. (A) Representative immunostaining images of mouse DRG neurons using ST2 antibody alone (Control) or ST2 antibody+blocking peptide (Peptide preabsorption). (B) Summary of the fluorescence intensities of neurons stained with ST2 antibody (Control) or ST2 antibody+blocking peptide (Peptide preabsorption). A total of 65 and 68 neurons were included in each group, respectively. (Scale bar, 20 µm.) **P < 0.01. Student’s t test was used for the analysis.
Fig. S2.
Fig. S2.
Characterization of ST2 expression in DRG neurons and skin nerve fibers. (A) Immunostaining showing the overlapping of ST2 with the accessory subunit IL-1RAcP and TRPV1 that labels nociceptive sensory DRG neurons but not with GS that labels satellite glial cells. (B) Immunostaining showing the overlapping of ST2 with PGP9.5-positive nerve fibers in mouse skin. (Scale bars, 20 µm.)
Fig. 3.
Fig. 3.
IL-33 induces Ca2+ mobilization in cultured DRG neurons isolated from urushiol-challenged mice. (A) Pseudocolor Fura-2 ratiometric images of DRG neurons isolated from C1-T1 DRGs of mice challenged with urushiol. Images show Ca2+ responses of DRG neurons in control condition and upon IL-33 (1 µg/mL), CQ (300 µM), and KCl (40 mM) application. White arrows indicate neurons that responded positively to IL- 33. (Magnification: 10×.) (B) Time course traces illustrate the different types of Ca2+ responses upon IL-33 and CQ application: cell responding to both IL-33 and CQ, pink; cell responding to IL-33 only, red; cell responding to CQ only, blue; cell responding to neither IL-33 nor CQ, black. (C) Venn diagram showing the overlapping of IL-33–positive (IL-33+) with CQ-positive, histamine (Hist)-positive, mustard oil (MO)-positive, and capsaicin (Cap)-positive neuronal populations. Each Venn diagram contains 200–300 DRG neurons. A neuron was considered IL-33+ if the peak Ca2+ response was >20% of baseline. (D) Summary of percentages of DRG neurons responding to vehicle (Veh; 0.1% BSA) and IL-33 in control, Ca2+-free extracellular solution, ruthenium red (RR; 10 µM), HC-030031 (HC; 100 µM), AMG9810 (AMG; 6 µM), isotype control IgG (Iso IgG; 0.5 mg/mL), and ST2-neutralizing antibody (0.5 mg/mL)-treated conditions. A total of 6–12 fields of observation were included in each group (each group contains 300−800 neurons from three to five mice). *P < 0.05; **P < 0.01; ##P < 0.01 compared to IL-33, urushiol-challenged. Student’s t test or one-way ANOVA followed by Tukey post hoc test was used for statistical analysis.
Fig. 4.
Fig. 4.
Effects of inhibition of IL-33/ST2 signaling on chronic itch in urushiol-challenged mice. (A) Treatment of urushiol-induced mouse ACD model. Mice were treated (i.p.) with isotype control IgGs (Iso IgGs) or IL-33– or ST2-neutralizing antibody (Ab) every day for a total of five times. Scratching behaviors were monitored at 0-, 4-, and 24-h time points as indicated. (B and C) Summarized scratching behaviors of unchallenged mice (Ace) and urushiol-challenged mice (Uru) after the treatment with isotype control IgGs, IL-33–neutralizing (15 µg per mouse), or ST2-neutralizing (50 µg per mouse) antibody. (D) Motor coordination behavior measured by rotarod. Mice were treated with isotype control IgGs or IL-33, ST2 antibody. n = 7 or 8 mice per group. **P < 0.01; ##P < 0.01; NS, no significance. One-way ANOVA followed by Tukey post hoc test or Student’s t test was used for statistical analysis.
Fig. S3.
Fig. S3.
Effects of inhibition of IL-33/ST2 signaling on skin inflammation of urushiol-challenged mice. (A and B) Bifold thickness of neck skin measured daily. (C) TEWL measured at the neck skin 24 h after the last (fifth) neutralizing antibody or isotype control IgGs treatment. (D) Dermatitis score of urushiol-challenged mice treated with isotype control IgGs or IL-33– or ST2-neutralizing antibody. n = 7 or 8 mice per group. **P < 0.01; ##P < 0.01. One- or two-way ANOVA followed by Tukey post hoc test was used for statistical analysis.
Fig. 5.
Fig. 5.
IL-33 promotes itch-related scratching in urushiol-induced ACD mice through ST2. (A) Experimental scheme of urushiol-induced ACD on mouse neck. (B and C) Scratching behavior at 0 h (B) and 4 h (C) in unchallenged (acetone-treated; Ace) and urushiol-challenged (Uru) mice in 5-min intervals during a 30-min period after vehicle (0.1% BSA) or IL-33 (+IL-33; 300 ng per site) injection at the nape of the neck. (D) Summarized scratching behavior at 0- and 4-h time point for the entire 30-min recording period. (E) Scratching behaviors of urushiol-challenged mice upon vehicle (0.1% BSA), IL-33–neutralizing (300 ng per site), and IL-33+ST2–neutralizing antibody (ST2 Ab; 50 µg per site) injection at the nape of neck at 0- and 4-h time points. (F) Effects of IL-33 or IL-33/cetirizine coadministration on cheek-scratching behavior recorded within 30 min after the third urushiol challenge of the cheek. Acetone group was challenged with acetone only. Cetirizine (10 mg/kg) or PBS was administered (i.p.) 30 min before IL-33 injection. Vehicle 1, 0.1% BSA in PBS (Veh1); Vehicle2, PBS (Veh2). (G) Cheek-wiping behavior in the same first three groups of mice shown in F. n = 7 or 8 mice per group. *P < 0.05, **P < 0.01, ##P < 0.01. NS, no significance. One- or two-way ANOVA followed by Tukey post hoc test was used for statistical analysis.
Fig. 6.
Fig. 6.
Effects of DRG-specific knockdown of ST2 on the itch response of urushiol-induced ACD mice. (A) Protocol for i.t. delivery of ST2 siRNA or scrambled control siRNA to mice. (B and C) qPCR analysis of transcript levels of St2 (Il1rl1), Il1rl2, and Il31ra in DRGs (B) or spinal cords (C) of ST2 siRNA or scrambled siRNA treatment groups. (D) Immunostaining of DRG sections showing analyzing expression of ST2 protein of ST2 siRNA- or scrambled siRNA-treated group. (Magnification: 60×.) (E) Summary of ST2 staining fluorescence intensities of neurons from ST2 siRNA or scrambled siRNA-treated mice. A total of 100–120 neurons pooled from five mice from each group were compared. (F) Analysis of scratching behavior of urushiol-induced ACD mice after i.t. injection of ST2 siRNA. (G) Bifold skin thickness of urushiol-induced ACD mice treated with ST2 siRNA or scrambled siRNA. (H) Comparison of motor coordination activity of siRNA-treated mice tested with rotarod. n = 6 or 7 mice per group. *P < 0.05; **P < 0.01; ##P < 0.01. NS, no significance. Student’s t test or one-way ANOVA followed by Tukey post hoc test was used for statistical analysis.
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