Selective role of neurokinin B in IL-31–induced itch response in mice
Authors: Daiji Sakata, Takehito Uruno, Keisuke Matsubara, Tsugunobu Andoh, Kazuhiko Yamamura, Yuki Magoshi, Kazufumi Kunimura, Yasuhisa Kamikaseda, Masutaka Furue, Yoshinori Fukui
Journal: Journal of Allergy and Clinical Immunology (2019)

Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting 15% to 20% of the general population in developed countries.1Weidinger S. Beck L.A. Beiber T. Kabashima K. Irvine A.D. Atopic dermatitis.Nat Rev Dis Primers. 2018; 4: 1Crossref PubMed Scopus (127) Google Scholar It is characterized by recurrent eczematous legions and intense itch. Because the itch sensation induces scratching behavior, which exacerbates the skin inflammation and disturbs the quality of life of affected individuals, chronic itch is a challenging clinical problem in the treatment of AD. Itch can be induced by various chemical mediators. Among them, much attention has been paid to IL-31 as an AD-associated itch mediator since the discovery of the pruritogenic action of IL-31 in mice.2Dillon S.R. Sprecher C. Hammond A. Bilsborough J. Rosenfeld-Franklin M. Presnell S.R. et al.Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice.Nat Immunol. 2004; 5: 752-760Crossref PubMed Scopus (658) Google Scholar IL-31 is mainly produced by CD4+ T cells and transmits the signals via a heterodimeric receptor composed of IL-31 receptor A and oncostatin M receptor (OSMR), both of which are expressed in various cell types including dorsal root ganglion (DRG) neurons.3Cevikbas F. Wang X. Akiyama T. Kempkes C. Savinko T. Antal A. et al.A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: involvement of TRPV1 and TRPA1.J Allergy Clin Immunol. 2014; 133: 448-460Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar A recent clinical study has demonstrated that blockade of IL-31 signals by a specific antibody for IL-31 receptor A alleviates pruritus in patients with AD.4Ruzicka T. Hanifin J.M. Furue M. Pulka G. Mlynarczyk I. Wollenberg A. et al.Anti-interleukin-31 receptor A antibody for atopic dermatitis.N Engl J Med. 2017; 376: 826-835Crossref PubMed Scopus (308) Google Scholar However, the neuronal mechanism underlying IL-31–induced itch sensation is poorly understood. Mutations of DOCK8 in humans cause a combined immunodeficiency characterized by AD. We have previously reported that DOCK8-deficient (Dock8–/–), but not Dock8+/–, mice spontaneously develop AD-like skin disease when crossed with transgenic mice expressing AND T-cell receptor (designated AND Tg mice). In this model, the disease manifestations were completely lost when either OSMR or IL-31 was deleted (see Fig E1 in this article's Online Repository at www.jacionline.org). To identify candidate molecules that mediate IL-31–induced itch sensation, we performed microarray analysis of the DRG neurons and found that 698 genes were expressed at higher levels in Dock8–/– AND Tg mice than in Dock8+/– AND Tg mice (see Table E1 in this article's Online Repository at www.jacionline.org), the second highest of which was Tac2 encoding neurokinin B. To confirm the microarray data, we performed real-time PCR analyses. Although the expression of Il31ra was comparable between Dock8–/– AND Tg and Dock8+/– AND Tg littermates, the expression of Tac2 in the DRG neurons increased 23-fold in Dock8–/– AND Tg mice, which was also canceled by deleting OSMR or IL-31 (Fig 1, A; see Fig E2 in this article's Online Repository at www.jacionline.org). Immunohistochemical analyses of Dock8–/– AND Tg mice revealed that neurokinin B was expressed by the IL-31 receptor A+ DRG neurons (Fig 1, B). Neurotransmitters are stored in synaptic vesicles and released on stimulation. Indeed, neurokinin B was released in vitro from primary DRG neurons of wild-type (WT; C57BL/6) mice in response to IL-31 (Fig 1, C). However, the release of neurokinin B was completely abolished when primary DRG neurons lack OSMR expression (Fig 1, C). Thus, neurokinin B is induced in and released from DRG neurons in response to IL-31. To examine the physiological significance of neurokinin B in IL-31–induced itch sensation, we developed 2 lines of Tac2-deficient (Δ4 and Δ15) mice by using the CRISPR/Cas9 nuclease system (see Fig E3 in this article's Online Repository at www.jacionline.org). Intradermal injection of histamine, chloroquine, and protease-activated receptor 2 agonist (SLIGRL-NH2) comparably induced scratching in both Tac2–/– (Δ4) and Tac2+/– mice (Fig 1, D), as was previously reported.5Mar L. Yang F.C. Ma Q. Genetic marking and characterization of Tac2-expressing neurons in the central and peripheral nervous system.Mol Brain. 2012; 5: 3Crossref PubMed Scopus (24) Google Scholar However, although IL-31 administration induced scratching in Tac2+/– mice, Tac2–/– (Δ4) mice were significantly less sensitive to IL-31 stimulation (Fig 1, D). Similar results were obtained when Tac2–/– mice (Δ15) were analyzed (see Fig E4 in this article's Online Repository at www.jacionline.org). Interestingly, Tac2 deficiency markedly improved the skin inflammation and scratching behavior in Dock8–/– AND Tg mice without affecting serum IL-31 levels (Fig 1, E-G). Thus, neurokinin B is selectively required for transmission of IL-31–induced itch sensation. Many pruritogens require natriuretic polypeptide b (Nppb) and gastrin-releasing peptide (GRP) to transmit itch sensation in the spinal cord.6Bautista D.M. Wilson S.R. Hoon M.A. Why we scratch an itch: the molecules, cells and circuits of itch.Nat Neurosci. 2014; 17: 175-182Crossref PubMed Scopus (205) Google Scholar To examine whether IL-31 uses GRP or Nppb to transmit itch sensation in the spinal cord, we specifically ablated neurons expressing GRP receptor or Nppb receptor by intrathecally injecting toxin (saporin)-conjugated GRP or Nppb, respectively. Although Nppb has been implicated in IL-31–mediated skin inflammation in the periphery,7Meng J. Moriyama M. Feld M. Buddenkotte J. Buhl T. Szöllösi A. et al.New mechanism underlying IL-31–induced atopic dermatitis.J Allergy Clin Immunol. 2018; 141: 1677-1689Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar IL-31–induced scratching was unaffected by intrathecal injection of Nppb-saporin (Fig 2, A). In contrast, treatment with GRP-saporin reduced IL-31–induced scratching (Fig 2, A). The Grp expression in the DRG neurons increased 4.5-fold in Dock8–/– AND Tg mice as compared with that in Dock8+/– AND littermates (see Fig E2). Although GRP was released in vitro from WT DRG neurons in response to IL-31, GRP release was hardly detected when primary DRG neurons from Tac2–/– (Δ4) mice were similarly stimulated (Fig 2, B). In addition, itch response induced by intrathecal injection of neurokinin B was lost when neurons expressing GRP receptor were ablated beforehand by toxin treatment (Fig 2, C). Neurokinin B transmits the signal through neurokinin 3 receptor (NK3R), a G protein–coupled tachykinin receptor. Immunohistochemical analyses of Dock8–/– AND Tg mice revealed that GRP was expressed by the NK3R+ DRG neurons and neurokinin B+ DRG neurons (Fig 2, D). These results indicate that neurokinin B acts upstream of GRP to transmit IL-31–induced itch sensation (Fig 2, E). So far, several selective antagonists for NK3R such as osanetant and fezolinetant have been developed.8Spooren W. Riemer C. Meltzer H. NK3 receptor antagonists: the next generation of antipsychotics?.Nat Rev Drug Discov. 2005; 4: 967-975Crossref PubMed Scopus (104) Google Scholar, 9Fraser G.L. Ramael S. Hoveyda H.R. Gheyle L. Combalbert J. The NK3 receptor antagonist ESN364 suppresses sex hormones in men and women.J Clin Endocrinol Metab. 2016; 101: 417-426Crossref PubMed Scopus (43) Google Scholar When osanetant was intraperitoneally injected into WT mice, IL-31–induced scratching was significantly attenuated (Fig 2, F). Similar results were obtained when fezolinetant was administered orally (see Fig E5 in this article's Online Repository at www.jacionline.org). In contrast, treatment with NK3R antagonists failed to suppress itch response induced by histamine, chloroquine, and SLIGRL-NH2 (Fig 2, F; see Fig E5). Thus, pharmacological inhibition of NK3R selectively attenuates IL-31–induced itch sensation. Although NK3R antagonists suppress sex hormones by modulating gonadotropin secretion, its effect is transient and reversible, with no major side effects reported as yet.8Spooren W. Riemer C. Meltzer H. NK3 receptor antagonists: the next generation of antipsychotics?.Nat Rev Drug Discov. 2005; 4: 967-975Crossref PubMed Scopus (104) Google Scholar, 9Fraser G.L. Ramael S. Hoveyda H.R. Gheyle L. Combalbert J. The NK3 receptor antagonist ESN364 suppresses sex hormones in men and women.J Clin Endocrinol Metab. 2016; 101: 417-426Crossref PubMed Scopus (43) Google Scholar Therefore, NK3R antagonists may be another option for treating AD-associated itch particularly in adults. Tac2–/– mice were developed by using the CRISPR/Cas9 genome editing system. A targeting site within the exon 4 of mouse Tac2 was selected using the CHOPCHOP web design tool (https://chopchop.rc.fas.harvard.edu/). Two complementary oligonucleotides (5′-CACCGAGTGCTGAGCAAGGCTAGCG-3′ and 5′-AAACCGCTAGCCTTGCTCAGCACTC-3′) containing the guide sequence (underlined) and Bbs I ligation adaptors were synthesized, annealed, and ligated into the Bbs I–digested px330 vector for coexpression of single guide RNA and Cas9 protein. The pX330 vector (5 ng/μL in Dulbecco's PBS) was injected into the pronuclei of in vitro fertilized eggs of C57BL/6 mice in M2 medium (Sigma-Aldrich, St Louis, Mo). The injected zygotes were cultured in CZB medium at 37oC, 5% CO2 until 2-cell stage embryos develop. Then, 24 to 36 embryos were transferred into the oviducts of pseudopregnant ICR female mice. Genotype of offspring mice was identified by genomic PCR using primers (5′-CTCTCCCCTACAAGGACTCTGA-3′ and 5′-CCAATCTAATCTTCAGAACGCC-3′) followed by TA cloning of the products and sequencing. The offspring mice carrying desired mutation (Δ4 and Δ15) were crossed with C57BL/6 mice or Dock8–/– AND Tg mice.E1Yamamura K. Uruno T. Shiraishi A. Tanaka Y. Ushijima M. Nakahara T. et al.The transcription factor EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL-31 induction.Nat Commun. 2017; 8: 13946Crossref PubMed Scopus (39) Google Scholar Dock8+/– AND Tg and Dock8–/– AND Tg mice with or without OSMR expression have been described previously.E1Yamamura K. Uruno T. Shiraishi A. Tanaka Y. Ushijima M. Nakahara T. et al.The transcription factor EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL-31 induction.Nat Commun. 2017; 8: 13946Crossref PubMed Scopus (39) Google Scholar For generation of Il31–/– mice, a targeting vector was designed on the basis of pNT1.1 vector to insert the gene encoding–enhanced green fluorescent protein and a flippase recognition target–flanked neomycin-resistant cassette (neo) immediately after the initiation codon and was introduced into embryonic stem cells by electroporation. Correctly targeted embryonic stem clones were microinjected into C57BL/6 blastocysts, and the male chimeras obtained were crossed with C57BL/6 female mice. Heterozygous mutant mice were crossed with CAG-FLPe transgenic mice (RBRC01843) to remove neo, and neo-deleted mutant mice were backcrossed with C57BL/6 mice for more than 5 generations before crossing with Dock8+/– AND Tg and Dock8–/– AND Tg mice. All mice were maintained under specific-pathogen-free conditions in the animal facility of Kyushu University, and age- and sex-matched littermate mice were used as controls. Dock8+/– AND Tg and Dock8–/– AND Tg mice with or without Osmr, Il31, and Tac2 were analyzed at age 12 to 18 weeks. Tac2–/– mice, Tac2+/– mice, and WT mice were analyzed at age 7 to 10 weeks. All animal experiments were conducted according to relevant national and international guideline contained in the “Act on Welfare and Management of Animals” (Ministry of Environment of Japan) and the “Regulation of Laboratory Animals” (Kyushu University). The protocol of animal experiments was approved by the Committee of Ethics on Animal Experiments of Kyushu University. Ablation of spinal cord neurons expressing GRP receptor or Nppb receptor was performed by intrathecal (segment L3/4) injection of GRP-saporin or Nppb-saporin, respectively (2 μg in 5 μL each, Advanced Targeting Systems, San Diego, Calif). As a control, nontargeted saporin (designated Blank) was also injected. Mice were used for experiments 2 weeks after toxin injection. Before experiments, mice were put into an acrylic cage (11 × 14 × 20 cm) for at least 1 hour for acclimation. Then, the itch-inducing substances dissolved in sterile saline were injected intradermally into the shoulder of the mice at a volume of 50 μL, and their behaviors were video-recorded using HDR-CX390 (Sony, Tokyo, Japan). Playback of the video was used for determination of the total number of scratching bouts per the specified times. When mice scratch, they stretch their hind paw toward the itchy spot, lean the head toward the hind paw, rapidly move the paw several times, and then lower it back to the floor. A series of these movements was counted as 1 bout of scratching. The following itch-inducing substances were used for intradermal injection in this study: IL-31 (1 μg in 50 μL; Peprotech, Rocky Hill, NJ), SLIGRL-NH2 (100 μg in 50 μL; BACHEM, Bubendorf, Switzerland), chloroquine (100 μg in 50 μL; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and histamine (100 μg in 50 μL; FUJIFILM Wako Pure Chemical Corporation). In some experiments, the NK3R antagonist osanetant (Axon Medchem, Groningen, The Netherlands) or fezolinetant (Haoyuan ChemExpress, Shanghai, China) was administered intraperitoneally or orally at concentration of 5 mg/kg or 10 mg/kg, respectively, 45 minutes before intradermal injection of itch-inducing substances. Skin tissues were fixed in 4% (w/v) paraformaldehyde and embedded in paraffin blocks. Sections (3 μm thick) were stained with hematoxylin and eosin, and examined by light microscopy. For immunofluorescence analyses of the DRG, mice were euthanized with isoflurane and subjected to perfusion with 4% paraformaldehyde in PBS. The DRG tissues were collected, postfixed overnight at 4°C, and cryoprotected overnight in 30% sucrose-PBS. Then, tissue samples were embedded in OCT compound (Sakura Finetech, Tokyo, Japan) and frozen on dry ice. After cryostat sections (10 μm thick) were prepared, they were blocked with G-Block (GenoStaff, Tokyo, Japan; GB-01) for 30 minutes at room temperature and incubated overnight at 4°C with primary antibodies. The staining was detected and visualized with fluorophore-conjugated secondary antibodies (Thermo Fisher, Waltham, Mass). 4′-6-Diamidino-2-phenylindole, dihydrochloride (DOJINDO, 1:5000) was used for nucleus staining. All images were obtained with a laser scanning confocal microscope (FV3000, Olympus, Tokyo, Japan). The following primary antibodies were used in this study: rabbit antibody for neurokinin B (Novus Biologicals, Centennial, Colo; NB300-201, 1:500), goat antibody for IL-31 receptor A (R&D Systems, Minneapolis, Minn; AF2107, 1:100), rabbit antibody for NK3R (Novus Biologicals; NB300-102, 1:50), and goat antibody for GRP (Santa Cruz Biotechnology, Santa Cruz, Calif; sc-7788, 1:100). Total RNA was isolated using ISOGEN (Nippon Gene, Tokyo, Japan), and cRNA was amplified and labeled using a Low Input Quick Amp Labeling Kit (Agilent Technologies, Santa Clara, Calif). The cRNA was then hybridized to a 44 K 60-mer oligomicroarray (Whole Mouse Genome oligo DNA Microarray Kit version 2.0; Agilent Technologies). The hybridized microarray slides were scanned using an Agilent scanner. The relative hybridization intensities and background hybridization values were calculated using Feature Extraction Software version 9.5.1.1 (Agilent Technologies). Raw signal intensities and flags for each probe were calculated from the hybridization intensities and spot information, according to the procedures recommended by Agilent Technologies. To identify up- or downregulated genes in experimental samples, we calculated Z scores and ratios from the normalized signal intensities of each probe (upregulated genes, Z score > 2.0 and ratio > 1.5-fold; downregulated genes, Z score < −2.0 and ratio < 0.66-fold). The microarray data that support the finding of this study are available in Gene Omnibus with the primary accession code GSE118986. Total RNA from each tissue was isolated using ISOGEN (Nippon Gene). After treatment with RNase-free DNase I (Life Technologies), RNA samples were reverse-transcribed with oligo (dT) primers (Life Technologies) and SuperScript III reverse transcriptase (Life Technologies) for amplification by PCR. The following primers were used for real-time PCR: for Tac2, 5′-TCGTGAAAGTGCTGAGCAAG-3′ and 5′-GTGTCTGGTTGGCTGTTCCT-3′; for Grp, 5′-CACGGTCCTGGCTAAGATGT-3′ and 5′-ATACAGGGACGGGGATTCAT-3′; for Il31ra, 5′-TCCTGAGGATCCCAGATGTC-3′ and 5′-GGAGCCACTCCACTATCCAA-3′; and for Hprt, 5′-CTGGTGAAAAGGACCTCTCG-3′ and 5′-TGAAGTACTCATTATAGTCAAGGGCA-3′. Real-time PCR was performed on CFX Connect Thermal Cycler (BIO-RAD, Hercules, Calif) using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, Calif). The expressions of mouse target genes were normalized to expression of Hprt gene. CFX Manager software (version 3.1) supplied with the instrument was used for analyses. Data are expressed as relative values to that of a sample from Dock8+/–Osmr+/+Il31+/+ AND Tg mice (Fig 1, A) or a sample from Dock8+/– AND Tg mice (Fig E2). DRG neurons were prepared from the specified mouse lines and stimulated in vitro with IL-31 (10 μg/mL; Peprotech). The culture supernatants were recovered 30 minutes after stimulation, and the concentrations of neurokinin B and GRP were measured with ELISA kits (MyBiosource, San Diego, Calif; MBS744693 for neurokinin B and RayBiotech, Norcross, Ga; EIAM-GRP for GRP), according to the manufacturers' instructions. Statistical analyses were performed using GraphPad Prism7 (GraphPad Software, La Jolla, Calif). The data were initially tested with a Kolmogorov-Smirnov test for normal distribution. Parametric data were analyzed using a 2-tailed unpaired Student t test when 2 groups were compared. Nonparametric data were analyzed with a Mann-Whitney test when 2 groups were compared. Data are expressed as mean ± SD, and P values of less than .05 were considered significant . The expression data on Tac2 gene encoding neurokinin B are indicated in boldface.